This manuscript is structured as follows: the first part consists in a bibliographic introduction, with the aim of providing a general overview of CO2 and its role in biology, and then focusing on [NiFe]-CODHs and its possible integration in biotechnological devices. A second section describes our findings on RrCODH and concern the characterization of the recombinant enzyme and its immobilization on functionalized carbon nanotubes, subsequently integrated in a functional device (Chapters 2 and 3).

Chapter 4 is dedicated to a preliminary study of electron transfer from the buried active site of RrCODH to an external partner, with the aim of understanding its mechanisms for its integration and possible future use in CO2RR devices. The last section will summarize the findings highlighted in the other chapters and provide future perspectives for the project (Chapter 5).

"Flattening the curve!"

This is a phrase that we are used to hearing lately, too bad that the latter is almost exclusively referring to the Covid-19 pandemic. Humanity is facing several problems including the one just mentioned, but this must not distract out attention from flattening the climate change curve. Climate change can be translated into two related themes: the warming of the earth due to human activity linked to excessive emissions of greenhouse gases and the resulting change in weather patterns (as ironically highlighted in Figure 0).

If we type one of the terms in bold above on any browser, we will find infinite content, but in all of them we will also find: carbon dioxide or CO2. This is because CO2 is the main actor of climate change, accounting for 80 % of the total composition of greenhouse gases, other gases being methane (10 %), nitrous oxide (7 %) and fluorinated gases (3 %).

Hence, greenhouse gases emissions can easily be expressed in CO2 emissions.

Consequently, the flattening of the climate change curve can be thought of as avoiding excessive CO2 emissions or in other words achieving net CO2 emissions of zero.

After this brief introduction, CO2 sounds like a very bad enemy to humanity. However, we are made of carbon and our life on earth will always involve the production and emission of CO2 into the atmosphere.

CO2 in biology doesn't sound so bad, since its metabolism plays a fundamental role as "building block" for the synthesis of organic molecules in living organisms. As I said above, every human activity as a cost of CO2, even the development and use of technologies with the aim of reducing CO2 emissions. Therefore, it is crucial to find cheaper alternatives, in terms of CO2 emissions.

As often observed in recent years, the use and development of technologies based on biological processes results in a better ecological impact on climate change. This is related to the fact that in biology there are no terms such as waste, garbage and trash, the latter terms being coined by the humans. What for us can be a waste, like the CO2 from our metabolism, represents a substrate for other organisms leading to the production of other products. As reported in the title of this thesis, our approach is based on the study and use of biological alternatives for CO2/CO inter-conversion Furthermore the attention will be focused on a biocatalyst in particular: the Carbon Monoxide Dehydrogenase (CODH).

CODH catalyzes the reversible reduction of CO2 to CO and represents an attractive biocatalyst in terms of activity, selectivity, ability to operate in mild conditions with low overpotentials. In addition, it does not require high production costs and uses earth abundant-metals. For these reasons, several studies have been conducted on [NiFe]-CODHs for applications in electro-catalytic CO2 reduction reaction (CO2RR). [NiFe]-CODHs consist of a complex multi-metallic NiFe4S4 active site, unique in biology, and additional FeS clusters are present to transfer electrons from the buried active site to the protein surface.

During my PhD, I have worked on the [NiFe]-CODH from Rhodospirillum rubrum (RrCODH). Historically this CODH has been isolated and characterized from the native bacterium in the early 80's but its studies have been limited due to the difficulties encountered during its production and purification. In this manuscript, I describe a new method for the heterologous production of a recombinant RrCODH in E. coli, which allows its production and purification in a single step with high yields and high specific activities towards the two carbonaceous substrates. Nowadays most of the energy is produced from fossil sources (Figure 1) [START_REF] Iea | Total primary energy supply (TPES) by source[END_REF]. This development model has improved the quality of human life but at the same time has brought to light severe problems such as the increase of carbon dioxide (CO2) emissions.

This has led to a reduction in air quality and mainly contributed to climate change with serious consequences on our eco system. Reducing CO2 emissions is currently a main objective of scientific and technological research. CO2 is the main carbon component of greenhouse gases and its atmospheric concentration increases at an average speed of 2.5 ppm per year (calculation based on the last ten years, 2010-2020) reaching 420 ppm in May 2021 [START_REF] Ed | Trends in Atmospheric Carbon Dioxide[END_REF]. Therefore, it is important to adopt a sustainable development model and move from the current political model. As the International Energy Agency (IEA) says, "The sustainable development scenario sets out the main changes that would be needed to achieve the key energy goals of the United Nations agenda for sustainable development". The sustainable development scenario of the IEA shows the possibility of reaching global "net zero" CO2 emissions in 2070, providing the reduction of air pollution and climate change [START_REF] Iea | Total primary energy supply (TPES) by source[END_REF]. As shown in Figure 2, the use of fossil resources must be reduced in favor of renewable resources. However nowadays, a radical change turns out to be utopian, so it is necessary to focus our attention on what can be implemented in our current economic model. (Current Policies) and announced targets (Sustainable Development) slow the growth of global CO2 emissions until 2040 but are not strong enough to force a peak in an expanding energy system. [START_REF] Iea | Total primary energy supply (TPES) by source[END_REF] This economic model introduces the concept of "closing the carbon cycle". At this point two questions spontaneously arise: what is the carbon cycle? What does it mean to close it? Carbon is the backbone of life on Earth. We are made of carbon, we eat carbon and our civilizations, our economies, our houses, our means of transport are built on carbon.

The carbon cycle is the natural way to circulate this element through different reservoirs with processes such as photosynthesis, respiration, weathering and fossilization (Figure 3). Today, changes in the carbon cycle are happening due to human activity. The latter is mainly related to the exploitation and combustion of fossil fuels. Then, carbon is released by combustion (or oxidation) into the atmosphere as CO2, and while the oceans and biomass grab about half of what we emit, the other stays in the atmosphere adding to the greenhouse effect [START_REF] Riebeek | Earth Observatory[END_REF] [START_REF] Styring | Preface[END_REF] [START_REF] Birdsey | Second State of the Carbon Cycle Report (SOCCR2)[END_REF]. As can be seen in Figure 3, the human effect increases net CO2 emissions year after year. Asterisks indicate that there is 95% confidence that the actual value is within 10% (*****), 25% (****), 50% (***), 100% (**), or >100% (*) of the reported value. (Adapted from [START_REF] Birdsey | Second State of the Carbon Cycle Report (SOCCR2)[END_REF] Hence, there are many actions that can be put in place to close the carbon cycle as well as achieve the reduction of CO2 emissions. Among them, the three main strategies are: decarbonization, carbon sequestration and carbon recycling.

Decarbonization is directly based on reduction or, at best, abolition the use of fossil fuels to switch to a renewable source. But this aim is difficult to achieve: firstly because renewable sources show limits in terms of scalability, versatility and storage time [START_REF] Nitopi | Progress and Perspectives of Electrochemical CO2 Reduction on Copper in Aqueous Electrolyte[END_REF]. Secondly, some sectors always require liquid fuels, such as transport.

Ultimately, some sectors, not related to energy, always involve CO2 emissions, such as intensive farming [START_REF] Nitopi | Progress and Perspectives of Electrochemical CO2 Reduction on Copper in Aqueous Electrolyte[END_REF].

The carbon sequestration, known as Carbon Capture and Storage (CCS) limits CO2 emissions into the atmosphere. The CCS involves the capturing of industrially produced CO2, compressing it for transportation and then injecting it underground (deep rock cavities, under sea and land) where it is permanently stored. In 2020, 26 large-scale CCS facilities are operating around the world which can capture and permanently store 40 Mtons of CO2 every year, representing less than 0.1 % of global carbon emissions (CCS Global Institute, 2020). The latter percentage is set to increase in the next years, with the development of 37 CCS facilities and the possibility of applying CCS facilities to every industrial plant (CCS Global Institute, 2020). The advantage of carbon sequestration is that, regardless of the source, fossil fuels or biomass for example, total net emissions are zero or negative in case of biomass. Unfortunately, the CCS facilities induce a price increase. For example, for a power plant the resulting energy displays an increase of more than 25% in the generated energy [START_REF] Nitopi | Progress and Perspectives of Electrochemical CO2 Reduction on Copper in Aqueous Electrolyte[END_REF]. Other problems are related to transportation and limited underground reservoirs.

The third strategy is based on the possibility to recycle or convert the CO2 into more reduced forms known as Carbon Capture and Utilization (CCU). The CCU is the most promising strategy, because it allows to reduce atmospheric CO2 and also to reduce the use of fossil fuels at the same time. The CO2 is a highly stable molecule and its conversion to other carbon-based molecules is difficult. However, several methods have been developed, such as chemical reduction, photochemical reduction, biological transformation and electrochemical reduction [START_REF] Duan | Metal-Free Carbon Materials for CO2 Electrochemical Reduction[END_REF]. Among these strategies, the electrochemical CO2 reduction reaction (CO2RR) allows to operate in mild conditions at ambient temperature and pressure. Furthermore, the accurate control of the applied potential, of the electrolyte and of the current allows the regulation of the reaction rates and products. In contrast, homogeneous reactions are based on indirect electrolysis, where the catalyst first accepts electrons from the electrodes and then donates electrons to the CO2. CO2RR shows several issues: the activation of the CO double bond is difficult due to its short bond length, the electron affinity of CO2 is low, and there is a large energy gap between the highest occupied and lowest unoccupied molecular orbitals [START_REF] Liang | Recent Development of Electrocatalytic CO2 Reduction Application to Energy Conversion[END_REF]. Furthermore, some side reactions can reduce the efficiency of the reaction, such as the hydrogen evolution reaction (HER) [START_REF] Liang | Recent Development of Electrocatalytic CO2 Reduction Application to Energy Conversion[END_REF]. When the goal is to apply CO2RR to energy conversion, the improvement of electrocatalysts is essential to overcome the mentioned drawbacks. Furthermore, an ideal CO2RR electrocatalyst should display a high selectivity combined to a strong CO binding energy and weak H binding energy [START_REF] Duan | Metal-Free Carbon Materials for CO2 Electrochemical Reduction[END_REF]. CO2RR technologies are studied and developed for two types of energy conversion: metal-CO2 based batteries and CO2 electroreduction to fuels.

CO2 can be electroreduced to a variety of carbonaceous products, such as carbon monoxide (CO), methane, formic acid, ethylene, ethanol, methanol and others (Figure 4).

Figure 4 Main valuable commodity chemicals from the CO2RR

When the goal is a large-scale production, selectivity and high yield are important factors. Among all the products mentioned above, CO is the most promising for several reasons. As a gas, CO directly allows its selective production and also does not require separation processes from the electrolyte solution. CO is an important "building block" in several industrial sectors, with a wide range of applications. The main uses of CO include the synthesis of acetic acid by catalytic carbonylation of methanol and formic acid by hydrolysis of methyl formate [START_REF] Küngas | Review -Electrochemical CO2 Reduction for CO Production : Comparison of Low-and High-Temperature Electrolysis Technologies Review -Electrochemical CO 2 Reduction for CO Production : Comparison of Low-and High-Temperature Electrolysis Technologies[END_REF]. Other applications include the production of various chemicals such as: acrylic acid, propanoic acid, acetic anhydride and dimethylformamide [START_REF] Küngas | Review -Electrochemical CO2 Reduction for CO Production : Comparison of Low-and High-Temperature Electrolysis Technologies Review -Electrochemical CO 2 Reduction for CO Production : Comparison of Low-and High-Temperature Electrolysis Technologies[END_REF]. In metallurgy, CO is used to reduce oxides to metals in blast furnaces and to produce highly pure Ni, Fe, Co, Cr, W and Mo via the metal carbonyl pathway [START_REF] Küngas | Review -Electrochemical CO2 Reduction for CO Production : Comparison of Low-and High-Temperature Electrolysis Technologies Review -Electrochemical CO 2 Reduction for CO Production : Comparison of Low-and High-Temperature Electrolysis Technologies[END_REF]. CO is also used as the main constituent of synthesis gas (syngas) along with H2. Syngas is used at very large scale for the production of liquid fuels and commodity products, such as: ammonia, methanol, mixed alcohols and others [START_REF] Chan | Recent progress on CO-rich syngas production via CO2 gasi fi cation of various wastes : A critical review on ef fi ciency , challenges and[END_REF] [START_REF] Drahansky | Syngas Production, Properties, and Its Importance[END_REF] (Alfano & Cavazza, 2018). Most CO2RRs are endergonic processes and require energy input. To give an idea of the energy required, Table 1 shows the standard potentials of thermodynamic reduction in aqueous solution at pH 7.0 [START_REF] Schlager | Biocatalytic and Bioelectrocatalytic Approaches for the Reduction of Carbon Dioxide using Enzymes[END_REF].

Table 1 Theoretical formal reduction potentials (E 0, ) of CO2 reduction to several products. The potentials are referred for aqueous solutions at pH 7.0 (Adapted from [START_REF] Schlager | Biocatalytic and Bioelectrocatalytic Approaches for the Reduction of Carbon Dioxide using Enzymes[END_REF] Reaction E 0, [V vs. NHE]

CO2

+ 1 e - → CO2 To perform any CO2 reduction process shown in Table 1 it is essential to take into account the energy barrier, which in the practical approach also means considering the overpotential of the reaction. To reduce these energy barriers, specific thermodynamic conditions are required, such as high pressures and/or high temperatures (above 300 °C) [START_REF] Schlager | Biocatalytic and Bioelectrocatalytic Approaches for the Reduction of Carbon Dioxide using Enzymes[END_REF]) (Ünlü et al., 2021) which result in unprofitable amounts of energy. In addition, conventional CO2RR methods also show poor selectivity and low yield (Ünlü et al., 2021). Furthermore, the catalyst should be able to carry out the reaction with the lowest overpotential in other words as close as possible to the standard reaction potential. All of these strategies always require a source of electrons.

The latter can have a CO2 cost if it comes from non-renewable energy. An ideal sustainable strategy for CO2RR should use a renewable source of energy provided by solar irradiation, wind, hydroelectric or geothermal energy. For these reasons, bioinspired materials such as models for artificial photosynthesis and other biological approaches have raised research interests in recent decades. Several bioinspired approaches for CO2RR have recently been developed, based on the direct use of biocatalysts such as enzymes or microorganisms (Y. [START_REF] Chen | Influence of gas management on biochemical conversion of CO2 by microalgae for biofuel production[END_REF] (Ünlü et al., 2021) [START_REF] Anwar | CO2 utilization: Turning greenhouse gas into fuels and valuable products[END_REF]. For example, microalgae (such as Nannochloropsis genus) can be used for the production of biofuels, in this case the microorganism can be genetically modified or evolved to increase the production of the desired products [START_REF] Maréchal | Carburants à base d 'algues oléagineuses Principes , filières , verrous[END_REF]. For now, these processes are generally on a laboratory scale, but some have been exploited at the industrial scale (e.g., LanzaTech).

Compared to other catalytic systems, biocatalysts show a very high selectivity and catalytic efficiency [START_REF] Schlager | Biocatalytic and Bioelectrocatalytic Approaches for the Reduction of Carbon Dioxide using Enzymes[END_REF] [START_REF] Anwar | CO2 utilization: Turning greenhouse gas into fuels and valuable products[END_REF]. Being a natural process, the reactions are carried out in mild conditions such as ambient temperature (20-30 °C), atmospheric pressure and aqueous solvents (Ünlü et al., 2021). Moreover, the catalyzed reactions are reversible. For these reasons the use of enzymes for CO2RR is interesting, in particular the use of dehydrogenases (Figure 5). Moreover, multiple enzymes can be coupled to perform multistep reactions (Figure 5B). For all the purposes listed above, it is critical to deeply characterize these biocatalysts, such as their role in native microorganisms, electron donor partners and catalytic

properties (e.g., optimal pH and temperature). The following paragraphs will focus on CO2/CO metabolism in microorganisms suitable as a model for developing bioinspired CO2RR processes.

A B

The microorganisms capable of metabolizing CO/CO2, therefore interesting for the bioinspiration or mimicry of CO2RR, all belong to the class of autotrophic organisms. All living forms of our planet can be distinguished in two categories based upon how they obtain their carbon sources: heterotrophs or autotrophs. Autotrophs are also known as primary producers because they are able to produce their own food from raw materials, such as CO2 and H2O, and energy. The organisms that belong to this category are plants, algae and some classes of bacteria and archaea. On the other hand, heterotrophs are known as consumers because they consume primary producers or other consumers, for example animals, fungi and many bacteria. This brief description and classification of living organisms should lead us to reflect on how the permanence of humans on the earth is strictly dependent on autotrophic microorganisms.

As shown in Figure 3, a key step in the global carbon cycle is the fixation of CO2 into organic carbon-based molecules. The microbial CO2 fixation proceeds through different pathways and different sources of energy. The latter allow to distinguish two types of autotrophs: photoautotrophs that like plants are able to harvest solar energy and chemoautotrophs where the energy source are reduced compounds such as hydrogen, sulfite, nitrite and other [START_REF] Nybo | Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals[END_REF]. To date, six natural carbon fixation pathways have been discovered (Figure 6)(F. [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]: the Calvin-Benson-Bassham cycle (CBB cycle), the reductive tricarboxylic acid cycle (rTCA cycle), the Wood-Ljungdahl pathway (WL pathway), the 3-hydroxypropionate bicycle (3HP bicycle), the dicarboxylate/4-hydroxybutyrate cycle (di-4HB cycle) and the 3-hydroxypropionate-4hydroxybutyrate cycle (3HP-4HB cycle).

The CBB cycle discovered in the 1940s is the best known and characterized carbon fixation pathway [START_REF] Calvin | The Path of Carbon in Photosynthesis[END_REF]. About 90 % of the natural CO2 fixation, carried out by microorganisms, algae and plants, uses this pathway (F. [START_REF] Gong | An Economy Based on Carbon Dioxide and Water[END_REF]. In short, the CBB cycle allows for the conversion of three CO2 into one molecule of glyceraldehyde-3-phosphate using nine molecules of ATP and six molecules of NADPH.

Compared to the other five pathways this is the most expensive in term of energy (Figure 6). [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF] The rTCA cycle consumes two molecules of CO2 to synthetize one molecule of Acetyl coenzyme A (Acetyl-CoA), using two molecules of ATP and four molecules of NAD(P)H (Figure 7). This pathway has been found in some anaerobic bacteria and photosynthetic green sulfur bacteria (F. [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]. [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF] The WL pathway is the only non-cyclic pathway, mainly present in anaerobic acetateproducing microorganisms (F. [START_REF] Gong | An Economy Based on Carbon Dioxide and Water[END_REF]. The energy source is the hydrogen and it converts two molecules of CO2, or one molecule of CO2 and one molecule of CO into one molecule of Acetyl-CoA, using one molecule of ATP and four of NAD(P)H (Figure 8). (Adapted from F. [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF] The 3HP bicycle found in non-sulfur green photosynthetic bacteria (F. [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF], uses three bicarbonate ions to synthetize one molecule of pyruvate and requires five molecules of ATP and five of NAD(P)H (Figure 9). (Adapted from F. [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF] The 3HP-4HB discovered in 2007 in some archaea use both hydrogen and sulfur as energy sources [START_REF] Berg | A 3-hydroxypropionate/4hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea[END_REF]. Each cycle involves two bicarbonate ions to synthetize one molecule of Acetyl-CoA, consuming four molecules of ATP and four molecules of NAD(P)H (Figure 10). (Adapted from F. [START_REF] Gong | Synthetic biology for CO2 fixation[END_REF] The di-4HB reported in 2008 is the latest discovered CO2 fixation pathway [START_REF] Huber | A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum <em>Ignicoccus hospitalis</em&gt[END_REF]. Present in anaerobic microbes, driven by hydrogen and sulfur, it uses a molecule of CO2 and a bicarbonate ion to synthetize one molecule of Acetyl-CoA by consuming three molecules of ATP and three molecules of NAD(P)H (Figure 11). [START_REF] Evans | A new ferredoxin-dependent carbon reduction cycle in a photosynthetic bacterium[END_REF] WL pathway 2CO2 → Acetyl-CoA 1 ATP, 4 NAD(P)H H2 [START_REF] Schulman | Total synthesis of acetate from CO 2 . V. Determination by mass analysis of the different types of acetate formed from 13 CO 2 by heterotrophic bacteria[END_REF] 3HP bicycle 3HCO3 - → Pyruvate 5 ATP, 5 NAD(P)H Light (M. C. [START_REF] Evans | A new ferredoxin-dependent carbon reduction cycle in a photosynthetic bacterium[END_REF] 3HP-4HB cycle CO2 + HCO3 -→ Acetyl-CoA 3 ATP, 4 NAD(P)H H2 and sulfur [START_REF] Berg | A 3-hydroxypropionate/4hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea[END_REF] di-4HB cycle 2HCO3 - → Acetyl-CoA 4 ATP, 4 NAD(P)H H2 and sulfur [START_REF] Huber | A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum <em>Ignicoccus hospitalis</em&gt[END_REF] After this brief description of the microbial CO2 metabolisms the attention will now be focused on a specific class of autotrophs microorganisms that can metabolize CO. The term carboxydotrophs has been coined to indicate these microbial organisms [START_REF] Meyer | Biology of aerobic carbon monoxide-oxidizing bacteria[END_REF]. These microorganisms are able to interconvert CO and CO2 according to the so-called Water-Gas Shift Reaction (WGSR):

CO + H2O ↔ CO2 + 2H + + 2e -

Carboxydotrophs, such as archaea, aerobic and anaerobic bacteria, are able to use CO as sole source of energy. In addition, some of them may also use CO as sole carbon source [START_REF] Robb | Life on the fringe: microbial adaptation to growth on carbon monoxide[END_REF]. This process is made such by the ubiquitous presence of the enzyme carbon monoxide dehydrogenase (CODH, CO oxidoreductase). CODH is widely conserved and distributed among aerobic and anaerobic microorganisms that are physiologically and phylogenetically different and presumably represents a very ancient metabolic activity.

CODH has the function of oxidizing CO, synthesizing acetyl-CoA (as in the WL pathway) or cleave it down into a variety of energy-producing pathways. The details on the different type of CODHs will be described in the following section, for now its role in CO metabolism in microorganisms will be presented.

It is noteworthy that in recent years, more and more aerobic CO oxidizing bacteria have been discovered and isolated, including Actinobacteria, Firmicutes and α-Proteobacteria (Table 3). These bacteria are unusual carboxydotrophs, as they prefer organic substrates as carbon and energy sources. However, they are still able to grow in the presence of high concentrations of CO (> 10%) [START_REF] King | Distribution, diversity and ecology of aerobic COoxidizing bacteria[END_REF]. In these bacteria the reducing equivalents resulting from the oxidation of CO are channeled through a respiratory chain insensitive to CO composed of ubiquinone and cytochromes, which ultimately lead to the reduction of oxygen [START_REF] Tiquia-Arashiro | Thermophilic Carboxydotrophs and Their Applications in Biotechnology. Chapter 2[END_REF], or nitrates [START_REF] Oelgeschläger | Carbon monoxide-dependent energy metabolism in anaerobic bacteria and archaea[END_REF]. In some aerobic carboxydotrophs, the energy conserved by CO metabolism can be used to fix CO2 to biomass through the CBB cycle [START_REF] Tiquia-Arashiro | Thermophilic Carboxydotrophs and Their Applications in Biotechnology. Chapter 2[END_REF]. These aerobic carboxydotrophs possess an aerobic CODH, phylogenetically different from anaerobic CODHs. The main difference is the metal content of the active site:

anaerobic CODHs uses metals available in anoxic atmosphere, such as nickel and iron, and are highly sensitive to oxygen inactivation. Differently, the aerobic CODHs use metals such as copper and molybdenum (Figure 12) which have become more bioavailable in oxic atmosphere. Other differences between the aerobic and anaerobic

CODHs are the biologic roles as well as their oligomeric composition of the subunits. Furthermore, the aerobic CODHs only catalyze the CO oxidation to CO2, with a specific activity of 23.2 U•mg -1 (Holger [START_REF] Dobbek | Catalysis at a dinuclear [CuSMo(=O)OH] cluster in a CO dehydrogenase resolved at 1.1-Å resolution[END_REF], while the anaerobic ones catalyze

B C A

Properties of some anaerobic carboxydotrophs are given in Table 4 (Sokolova et al., 2009) [START_REF] Diender | Pathways and Bioenergetics of Anaerobic Carbon Monoxide Fermentation[END_REF] [START_REF] Robb | Life on the fringe: microbial adaptation to growth on carbon monoxide[END_REF]. These respiratory processes include: hydrogenogenesis, desulfurization, acetogenesis and methanogenesis (Tiquiaarashiro, 2014). [START_REF] Diender | Pathways and Bioenergetics of Anaerobic Carbon Monoxide Fermentation[END_REF] [START_REF] Robb | Life on the fringe: microbial adaptation to growth on carbon monoxide[END_REF] Acetogens and methanogens possess mainly bifunctional CODH/ACS involved in the WL pathway [START_REF] Robb | Life on the fringe: microbial adaptation to growth on carbon monoxide[END_REF]. Sulfate reducers couple the oxidation of CO to CO2

with the reduction of sulfate. The latter is used as a terminal electron acceptor of the respiratory chain, thus producing sulfide [START_REF] Marietou | Sulfate Transporters in Dissimilatory Sulfate Reducing Microorganisms : A Comparative Genomics Analysis[END_REF]. Hydrogenogens includes bacteria such as Rhodospirillum rubrum and Carboxydothermus hydrogenoformans, which are able to grow by converting CO to CO2 and subsequently reduce protons to H2 derived from H2O. This process is known as the biological WGSR [START_REF] Fox | Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme[END_REF] [START_REF] Fox | Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum[END_REF] [START_REF] Soboh | Purification and catalytic properties of a CO-oxidizing: H2-evolving enzyme complex from Carboxydothermus hydrogenoformans[END_REF], is performed by a membrane associated multiprotein complex (Figure 14). Involving the monofunctional CODH for the oxidation of CO to CO2 from which the electrons are transferred through a ferredoxin (called CooF) to an Energy-Conserving Hydrogenase (ECH), which ultimately reduce protons to H2. This process is coupled to the translocation of protons across the cytoplasmic membrane leading to the formation of ATP by an ATP-synthase (Figure 14). [START_REF] Munk | Complete genome sequence of Rhodospirillum rubrum type strain (S1 T)[END_REF]. Its length is 3-10 µm, with a width of 0.8-1.0 µm. R. rubrum prefers to grow in habitats with a pH of 6.8-7.2 [START_REF] Bergey | Bergey's manual of determinative bacteriology[END_REF]. Its optimal growth temperature is 22-35 degrees Celsius [START_REF] Weaver | Temperature-sensitive mutations of the photosynthetic apparatus of Rhodospirillum rubrum[END_REF]. Under aerobic conditions the cells are colorless, and photosynthesis is Bergey's Manual Trust 1995) repressed. With a lower oxygen concentration, it is able to grow through an alcoholic fermentation. Under anaerobic conditions it can grow photosynthetically using bacteriochlorophyll which allows the absorption of light at wavelengths longer than those absorbed by plants (near infrared) (Christine [START_REF] Munk | Complete genome sequence of Rhodospirillum rubrum type strain (S1 T)[END_REF]. Under these conditions the colonies are purple because the cells express the carotenoid pigment necessary to gather the energy of light for photosynthesis. Furthermore, R. rubrum does not produce oxygen, but elemental sulfur as a by-product of photosynthesis (Christine [START_REF] Munk | Complete genome sequence of Rhodospirillum rubrum type strain (S1 T)[END_REF]. R. rubrum is a well-established model organism for studies on nitrogen fixation and the organism possesses two related but distinct nitrogenase systems that utilize a MoFe-and Fe-nitrogenases [START_REF] Lehman | Identification of an alternative nitrogenase system in Rhodospirillum rubrum[END_REF]. R. rubrum has a great interest in scientific research and biotechnological applications, including its capacity of producing biodegradable plastic. In fact, when the bacterium grows on CO the CO2 produced via the biological WGSR can be accumulated into cell material and poly(3-hydroxybutyrate) (PHB) (Figure 16). PHB is a polyhydroxyalkanoate (PHA), the latter are polyesters synthesized by many bacteria as energy and carbon storage molecules [START_REF] Revelles | Carbon roadmap from syngas to polyhydroxyalkanoates in Rhodospirillum rubrum[END_REF]. [START_REF] Revelles | Carbon roadmap from syngas to polyhydroxyalkanoates in Rhodospirillum rubrum[END_REF].

These polymers are biodegradable plastic materials that offer an alternative to petroleum-derived plastic, in addition the monomers can be combined within this family giving life to materials that make these bioplastics suitable for different commercial applications, such as biomedical implants or packaging articles [START_REF] Revelles | Carbon roadmap from syngas to polyhydroxyalkanoates in Rhodospirillum rubrum[END_REF].

For example, R. rubrum was cultivated in a bioreactor fed with CO and acetate, with a maximum yield of the PHB content for biomass of 30% w/w [START_REF] Karmann | Fed-batch cultivations of rhodospirillum rubrum under multiple nutrient-limited growth conditions on syngas as a novel option to produce poly(3-hydroxybutyrate) (PHB)[END_REF].

R. rubrum has also provided information on radiation resistance. The European Space

Agency is currently working on "MELiSSA life support system", a project for the development of numerous interconnected bioreactors inhabited by microorganisms and higher plants, with the aim of producing oxygen, water and food by recycling organic waste [START_REF] Mastroleo | Insight into the radiotolerance of the life support bacterium Rhodospirillum rubrum S1H by means of phenotypic and transcriptomic methods[END_REF]. This technology will be integrated in space, where it will be exposed to higher doses and different ionizing radiation spectra. Hence, it is necessary to screen the radiotolerance of the organisms that will be used. R. rubrum was subjected to sub-lethal doses of ionizing radiation, Cobalt-60 gamma rays, and specific marker genes for resistance to ionizing radiation were discovered and characterized [START_REF] Mastroleo | Insight into the radiotolerance of the life support bacterium Rhodospirillum rubrum S1H by means of phenotypic and transcriptomic methods[END_REF]. This may lead to advances in radiation resistance applied in research. Finally, R. rubrum is a hydrogenogenic bacterium and can be used in the field of renewable energies, in particular to upgrade synthesis gas (syngas).

Syngas is a mixture of gasses which, to date, derive mainly from the gasification of fossil fuels. Raw syngas are mainly composed of CO and H2, with a ratio of less than 0.6 (Alfano & Cavazza, 2018). Syngas represents a substrate for obtaining many chemicals, such as H2, ammonia, ethanol, methanol and other alcohols or liquid fuels via the Fischer-Tropsch processes [START_REF] Drahansky | Syngas Production, Properties, and Its Importance[END_REF] [START_REF] Spath | Preliminary Screening --Technical and Economic Assessment of Synthesis Gas to Fuels and Chemicals with Emphasis on the Potential for Biomass-Derived Syngas[END_REF]. To this end it is necessary to increase the H2/CO ratio (e.g., Fischer-Tropsch 4/1) and this is possible through the WGSR which is carried out industrially through inorganic metal catalysts and at high temperatures (350-500 °C) [START_REF] Nanda | Advancements and confinements in hydrogen production technologies[END_REF] [START_REF] Shafer | Fischer-tropsch: Product selectivity-the fingerprint of synthetic fuels[END_REF]. Najafpour, Younesi and collaborators explored different conditions for better fermentation of R.

rubrum and production of H2 from syngas. The best hydrogen production and production speed was 16 ± 1.1 mmol g -1 cell h -1 and 87 ± 2.4% at a fixed stirring speed of 500 rpm and a syngas flow rate of 14 ml/min, respectively [START_REF] Younesi | Biohydrogen production in a continuous stirred tank bioreactor from synthesis gas by anaerobic photosynthetic bacterium: Rhodopirillum rubrum[END_REF].

These systems can be attractive alternatives for the upgrading of syngas as well as for the production of H2. However, they are still at laboratory scale, because there are disadvantages such as the main represented by the gas/liquid mass transport. In addition, the high gas pressure can also limit or inhibit the microbial growth. For these reasons, it is essential to characterize and fully understand the chemistry behind these biological catalysts.

The metabolisms and microorganisms presented above have one factor in common: they all possess enzymes with cofactors containing transition metals, mainly iron and nickel. This common feature is directly related to the origin of life on earth. At the beginning of life, the earth was different from what it is now, the atmosphere was rich in gases such as H2, CO, CO2, N2 and CH4 [START_REF] Wächtershäuser | From volcanic origins of chemoautotrophic life to Bacteria, Archaea and Eukarya[END_REF]. These molecules were escaped from the interior of the Earth by magmatic or volcanic exhalations.

Furthermore, the presence of these events has made the oceans hot and well supplied with transition metals such as Fe(II) and Ni(II) [START_REF] Alfano | Structure, function, and biosynthesis of nickel-dependent enzymes[END_REF]. Under these environmental conditions, nickel could be considered as the fuel for the engine of life, the engine being the catalysts and the fuel the nickel as cofactor. These nickel-containing catalysts have allowed the development of the metabolism of microorganisms such as methanogenic archaea [START_REF] Alfano | Structure, function, and biosynthesis of nickel-dependent enzymes[END_REF]. These organisms can be considered as pioneers that triggered the complex evolution of life, because they have been able to promote the growth of the organic superstructure through carbon fixation, guided by inorganic substructures (catalysts with metal centers) [START_REF] Wächtershäuser | From volcanic origins of chemoautotrophic life to Bacteria, Archaea and Eukarya[END_REF]. The second most important evolutionary event was the change in the pioneering environment with a decrease in the volcano activity or magmatic fumes and consequent cooling of the upper mantle of the oceans. These geothermal events also reduced the nickel rich environment leading to a "nickel famine" drastically reducing the growth of methanogens [START_REF] Alfano | Structure, function, and biosynthesis of nickel-dependent enzymes[END_REF]. This environmental change of the earth is called the "great oxidation event" which has allowed the proliferation of other microorganisms, such as oceanic cyanobacteria. The latter developed photosynthetic metabolisms in which the final product was dioxygen, leading to a drastic change in the atmospheric composition. The appearance of oxygen in the atmosphere and the oxidative environment has induced the segregation of these ancestral microorganisms in anoxic niches where scientists have isolated them in recent centuries. [START_REF] Zambelli | Biochimica et Biophysica Acta Nickel impact on human health : An intrinsic disorder perspective[END_REF]. As mentioned earlier, nickel enzymes perform key functions in fundamental processes such as energy metabolism and virulence. These enzymes are either redox or non-redox.

Non-redox enzymes, such as urease (amidohydrolase), that catalyzes the hydrolysis of urea, use Ni(II) as a Lewis acid [START_REF] Mazzei | The Structure of the Elusive Urease-Urea Complex Unveils the Mechanism of a Paradigmatic Nickel-Dependent Enzyme[END_REF] As discussed above, redox Ni enzymes are mainly present in ancestral metabolisms of pioneer microorganisms. Another observation confirming this theory is that the NiFe active sites of Hydrogenase, CODH and ACS are coordinated to the protein via several cysteine residues that show structural affinities with natural metal sulfide catalysts (Figure 18) [START_REF] Nitschke | Biochimica et Biophysica Acta On the antiquity of metalloenzymes and their substrates in bioenergetics[END_REF]. In fact, these enzymes are defined as pre-Last

Universal Common Ancestor (pre-LUCA) catalysts [START_REF] Nitschke | Biochimica et Biophysica Acta On the antiquity of metalloenzymes and their substrates in bioenergetics[END_REF].

Figure 18 Diagram demonstrating the affinities between the natural sulfide nickelian mackinawite, greigite and violarite with the active centres of early metalloenzymes [NiFe]-H2ase, [NiFe]-CODH and acetyl coenzyme-A synthase, respectively. [START_REF] Alfano | Structure, function, and biosynthesis of nickel-dependent enzymes[END_REF].

In this section the focus will be on the two key enzymes involved in biological WGSR in between hydrogen metabolism and energy conservation, by coupling the exergonic electron transfer to the establishment of a transmembrane proton gradient (Δμ H + ) and are progenitors of complex I (NADH dehydrogenase) found in the respiratory chain [START_REF] Schoelmerich | Energy-converting hydrogenases: the link between H2 metabolism and energy conservation[END_REF]. In R. rubrum, the WGSR complex is encoded by two operons: cooMKLXUH and cooFSCTJ (Figure 19A). The cooMKLXUH operon encodes a CO-induced ECH. The cooFSCTJ operon encodes the ferredoxin (CooF), CODH (CooS) and related proteins (CooC, CooT and CooJ) for the insertion of nickel into the CODH [START_REF] Kerby | In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: Molecular and physiological characterization of cooCTJ[END_REF]. Present downstream of these two operons the cooA gene which encodes for the transcriptional regulator CooA. Its activation depends on CO and it induces the expression of the cooMKLXUH and cooFSCTJ operons [START_REF] Fox | Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum[END_REF]. CooA possesses a DNA binding domain and two heme domains.

The latter are responsible for the sensing and binding of CO, which induces an allosteric conformational change on CooA which allows interaction with the promoters of the CODH and ECH genes clusters [START_REF] Roberts | CooA, a paradigm for gas sensing regulatory proteins[END_REF]. In the true hydrogenogenic thermophilic C. hydrogenoformans, which grows in the presence of 100% CO as the only source of carbon and energy and produces equimolar quantities of H2 [START_REF] Svetlichny | Carboxydothermus hydrogenoformans gen. nov., sp. nov., a CO-utilizing Thermophilic Anaerobic Bacterium from Hydrothermal Environments of Kunashir Island[END_REF], these multiprotein complex is encoded by the cooA2CMKLXUH cluster and by the cooFS1 genes, interspaced by a 116-bp noncoding region (Figure 19B). [START_REF] Hedderich | Energy-converting [NiFe] hydrogenases: More than just H2 activation[END_REF].

The whole complex was purified [START_REF] Soboh | Purification and catalytic properties of a CO-oxidizing: H2-evolving enzyme complex from Carboxydothermus hydrogenoformans[END_REF] and it is formed by a homodimeric

Ni-containing CODH (CooS1), two ferredoxin (CooF) equipped with four iron sulfur clusters and a membrane bound [NiFe]-H2ase (CooMKLXUH) composed by four hydrophilic subunits and two membrane integral subunits (Figure 19C). This six subunits cluster that build the membrane-bound hydrogenase share similarity with subunits that form the catalytic core of energy-conserving NADH:quinone oxidoreductases (complex I) from E. coli [START_REF] Hedderich | Energy-converting [NiFe] hydrogenases: More than just H2 activation[END_REF]. The isolated complex showed maximal COoxidizing:H2-evolving activity of 450 U•mg -1 protein with 5% CO, higher concentration of CO inhibiting the hydrogenase [START_REF] Soboh | Purification and catalytic properties of a CO-oxidizing: H2-evolving enzyme complex from Carboxydothermus hydrogenoformans[END_REF]. Further, the complex also catalyzed the CO-and H2-oxidation which independently specific activity of 5 900 and 180 U•mg -1 , respectively [START_REF] Soboh | Purification and catalytic properties of a CO-oxidizing: H2-evolving enzyme complex from Carboxydothermus hydrogenoformans[END_REF].

The CODH:CooF:ECH complex coupled to the ATP-synthase may be the simplest form of respiration which combines CO/CO2 fixation with ATP synthesis in an ancestral pathway.

After this brief description of the ECHs, the second enzymatic partner of the biological WGSR, CODH will now be described in details.

[NiFe]-CODHs can be divided into four classes (Figure 20) [START_REF] Lindahl | The Ni-containing carbon monoxide dehydrogenase family: Light at the end of the tunnel?[END_REF] The ACDS complex catalyzes the transformation of three substrates: CO generated by the reduction of CO2 (CODH activity), a methyl group donated by a methylated B12 protein (corrinoid iron-sulfur protein or CFeSP, corresponding to the Y subunit) and coenzyme A (CoA) [START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF]. CoA reacts with CO and the methyl group to form acetyl-CoA, a source of energy and cellular carbon.

CO + CH3-CFeSP + CoA ↔ CH3-CO-CoA + CFeSP

The reaction is involved both in the fixation of CO/CO2 and in acetoclastic methanogenesis in the reverse direction. In this case, this reaction is used for the cleavage of acetyl-CoA to form CoA, methylated CFeSP and CO.

The reactions of synthesis and cleavage of the acetyl C-C and C-S bonds of acetyl-CoA are carried out at the active site called A-cluster (Figure 24A), located in the α subunit for ACS and β subunit for ACDS. The A-cluster is composed of a binuclear Ni-Ni site bridged with a cysteine thiolate to a [Fe4S4] cluster [START_REF] Gu | The A-Cluster in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Complex from Methanosarcina thermophila: Ni and Fe K-Edge XANES and EXAFS Analyses[END_REF]. The CODH activity in the ACDS complex is carried out by the α subunit, while β subunit for ACS. The crystal structure of the CODH component of the ACDS from Methanosarcina barkeri is available (W. [START_REF] Gong | Structure of the α2ε2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex[END_REF]. The structure revealed an oligomerization between two CODH (α) subunits and two ε subunits (α2ε2) (Figure 21AB). The α2ε2 oligomer shows a total of nine iron sulfur clusters (Figure 21C), in which each α subunit harbors a [Fe4S4] cluster The role of the ε subunits is unknown, it has been hypothesized that they can bind nucleotide cofactors and/or that they are involved in protein-protein interactions and electrons transfer with the other ACDS subunits [START_REF] Drennan | Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase[END_REF].

The CODH/ACS complex was purified from Moorella thermoacetica cultures and crystallized. Its crystal structural revealed an α2β2 oligomerization state (Figure 22), in which the central nucleus consists of two CODH subunits (β) and one ACS subunit (α) on each side of CODH.

ACS is made up of three main domains, the first one interacting with CODH via a ferredoxin interaction domain. The second domain is involved in the binding of CoA and the third domain contains the A-cluster. The latter domain undergoes structural rearrangements during the reaction, as shown by X-ray crystallography. Indeed, two

different conformations (open and closed states) are observed in the X-ray structure (Figure 22) [START_REF] Darnault | Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open α subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase[END_REF]. In addition, the CODH/ACS complex was crystallized in the presence of high-pressure xenon gas (Figure 23) [START_REF] Darnault | Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open α subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase[END_REF]. The crystal structure revealed the presence of 140 Å long gas tunnels that connect the CODH active site (C-cluster) to the ACS active site (A-cluster). The distance from these two active sites is 67 Å. The CO produced in the CODH subunit remains sequestered inside the enzyme and transferred to the ACS active site through these tunnels. They consist of a series of hydrophobic pockets in which the (Adapted from [START_REF] Darnault | Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open α subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase[END_REF] In the closed conformation of ACS subunit, the CO channel is open to allow the CO to reach A-cluster. However, in this configuration, the latter has no access to the physiological partner CFeSP. In the opened conformation, the A-cluster is rotated to allow the interface with CFeSP and the channel is blocked to prevent the release of CO.

During the formation of the acetyl-metal center intermediate, another closed conformation is necessary. Hence, A-cluster must be rotated to a more opened conformation to allow for CoA binding, thiolytic cleavage of the acetyl group and the formation and release of acetyl-CoA [START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF].

NiFeS clusters are present in both active sites of ACS (in subunit β of ACSD and subunit α of CODH/ACS) and CODH (in subunit α of ACSD and β subunit of CODH/ACS) (Figure 24A). In A-cluster there are two Ni atoms, namely the proximal Nip closest to the [4Fe4S] cluster and the distal Nid which is bridged to Nip through two cysteine thiolates and further coordinated by the two cysteine backbone amides. Nid is in square planar geometry and in the redox state 2+, while the Nip is coordinated by three S atoms and a labile position for substrate/product binding (a water molecule in the resting state), which completes a distorted square planar coordination [START_REF] Doukov | A Ni-Fe-Cu center in a bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase[END_REF].

The presence of the acetyl group in this position has been demonstrated with an enzyme in which Nip is replaced by Cu (Figure 24B) [START_REF] Doukov | A Ni-Fe-Cu center in a bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase[END_REF]. The enzyme containing Cu shows a lower activity than the native one, which demonstrates the importance of Ni in A-cluster. • ChCODH-I, like RrCODH, is the one involved in WGSR [START_REF] Svetlitchnyi | Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxideutilizing eubacterium Carboxydothermus hydrogenoformans[END_REF].

• ChCODH-II is associated with the membrane and its role would be related to the generation of NADPH [START_REF] Svetlitchnyi | Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxideutilizing eubacterium Carboxydothermus hydrogenoformans[END_REF].

• ChCODH-III belongs to the class III of [NiFe]-CODHs, and is part of a bifunctional ACS/CODH complex [START_REF] Wu | Life in hot carbon monoxide: The complete genome sequence of carboxydothermus hydrogenoformans Z-2901[END_REF].

• ChCODH-IV is found in a gene cluster close to an enzyme that reduces peroxide and shows a better oxygen tolerance than other CODHs. Hence, it was proposed to be involved in the response to oxidative stress via an O2/CO detoxification mechanism [START_REF] Domnik | CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2[END_REF].

• ChCODH-V has an unknown function. However, its X-ray structure shows differences in the organization of the iron-sulfur clusters compared to other monofunctional [NiFe]-CODHs [START_REF] Domnik | Biochemische und strukturelle Untersuchungen der Kohlenmonoxid-Dehydrogenasen CODH-II und CODH-IV aus Carboxydothermus hydrogenoformans[END_REF]. In fact, it shares similarities with Hybrid Cluster Proteins (HCPs) which contains a [Fe4S2O2] cluster. HCPs protect bacteria from nitrosative stress (J. [START_REF] Wang | The roles of the hybrid cluster protein, Hcp and its reductase, Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress[END_REF]. Furthermore, this enzyme cannot oxidize CO, therefore it may not be considered anymore as a CODH but rather as an HCP. However, an evolutionary link between the two families of enzymes is strongly suggested (Alfano, 2019).

To date, several structures of monofunctional 25A). The overall organization of the clusters is C-B'-D-B-C' (Fig. 25A), where the D-cluster bridges the two monomers. Each monomer is composed of three domains: a helical N-terminal domain, a central α/β domain (Rossmann-like) and a C-terminal α/β domain (Rossmann-like) (Figure 25B).

The two N-terminal domains contributes to the coordination of D-and B-clusters, via cysteine thiolates (Figure 26A), while the two Rossmann-like domains contribute to the coordination of C-cluster. [START_REF] Drennan | Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase[END_REF]. The C-cluster is a distorted [Ni3Fe4S] cubane coordinated to a mononuclear iron (Feu), unique in biology (Figure 26B). This cluster is coordinated by four cysteine thiolates (Cys338, Cys451, Cys481 and Cys531 in R. rubrum) and Feu is coordinated by the thiolate group of Cys300 (in R. rubrum) and the imidazole group of His265 (in R. rubrum). As mentioned above, the interconversion of CO/CO2 takes place at the C-cluster, according to:

CO + H2O ↔ CO2 + 2H + + 2e - CO oxidation yields CO2, protons and electrons, transferred from the buried active site to the protein surface, in order to reach the protein partners. B-clusters are the most likely candidates for electron transfer, due to their proximity to the active site (11 Å).

The role of the D-cluster is not entirely known and it will be discussed in Chapter 4. [START_REF] Spangler | Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum[END_REF] [START_REF] Bonam | Nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: in vivo and in vitro activation by exogenous nickel[END_REF]) [START_REF] Ensign | Nickel is required for the transfer of electrons from carbon monoxide to the iron-sulfur center(s) of carbon monoxide dehydrogenase from Rhodospirillum rubrum[END_REF]. This Ni-deficient CODH is inactive, but can be partially activated in vitro by adding nickel salt under reducing conditions [START_REF] Spangler | Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum[END_REF].

Furthermore, it also showed that the Feu atom is not present in the Ni-deficient enzyme and that the spectroscopic properties correspond to a classical [4Fe4S] cluster [START_REF] Spangler | Substitution of Valine for Histidine 265 in Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum Affects Activity and Spectroscopic States[END_REF]. As shown above, C-cluster is coordinated to the protein via five cysteines and one histidine residues (Figure 26), in particular Cys338, Cys451 and Cys481 coordinate the three iron of the [Ni3Fe4S] cluster, Cys531 coordinates the Ni atom and the Feu is coordinated by the His265 and Cys300 residues. In addition, Feu is linked to the cluster via a bridging sulfide. Site-directed mutagenesis was used to discover and understand the precise role of each residue in the electronic properties as well as in the biosynthesis of the C-cluster. Unexpectedly, when the coordinating Cys were mutated (Cys338Ala, Cys451Ala, Cys451Ser and Cys531Ala), the Ni content was comparable to that of the wildtype enzyme, although the CO oxidation activities were reduced (Table 5) [START_REF] Jeon | New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: Analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster[END_REF]. In contrast, The His265Val mutant exhibits reduced nickel content and drastically abolished activity (Table 5) [START_REF] Spangler | Substitution of Valine for Histidine 265 in Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum Affects Activity and Spectroscopic States[END_REF]. Ni content approximately twice as high as the native enzyme one (Table 5), suggesting that multiple Ni binding sites could be present.

These results has led to the proposal of a mechanism for the nickel insertion step (Figure 27) [START_REF] Jeon | New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: Analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster[END_REF]. Prior to the conversion of the (Wittenborn et al., 2019b). Taken together, these results suggest that Cys301 plays a role in the biogenesis of C-cluster, as well as in the Ni incorporation mechanism. All together these results led to the proposition of an alternative Ni insertion mechanism (Figure 30) (Wittenborn et al., 2019b). The physiological functions of CooC, CooT and CooJ, when present in CODH operons, have been demonstrated in R. rubrum (Figure 32) [START_REF] Kerby | Carbon monoxide-dependent growth of Rhodospirillum rubrum[END_REF] [START_REF] Kerby | In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: Molecular and physiological characterization of cooCTJ[END_REF].

First, R. rubrum in the absence of light is able to grow using CO as an energy source and is strictly dependent on CODH. The inactivation of the cooS gene hinders the growth of R. rubrum in the dark under CO (Figure 32). The same results were observed if the three cooC, cooT and cooJ genes are inactivated. However, in this latter case, a nickel supply to the growth medium compensates for the absence of the three genes. When only cooC gene is inactivated, R. rubrum cells require a nickel concentration 1000 times higher (650 µM) than wild type cells (0.5 µM) in order to grow in the dark. When the cooT and/or cooJ genes are inactivated, the cells require a nickel concentration of at least 50 times higher (better growth with 75 µM) than wild type cells.

However, in all cases, the CODH activity is not fully restored by Ni addition (at the most 50 % of the active enzyme), showing the importance of the three accessory proteins in CODH maturation and activation. However, the precise role of each of them is not yet clear: they could be either involved in the nickel delivery to C-cluster or in the trafficking and storage of intracellular nickel. In contrast, they were shown to be not involved in nickel uptake from the environment [START_REF] Watt | Ni2+ Transport and Accumulation in Rhodospirillum rubrum[END_REF]. [START_REF] Kerby | In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: Molecular and physiological characterization of cooCTJ[END_REF] CooC is an ATPase described as the major player in CODH maturation, similar to UreG and HypB which are GTPases involved for the maturation of urease and hydrogenase, respectively (Table 6) [START_REF] Jeon | Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum *[END_REF]. All CooC homologues have a strictly conserved CXC motif for the metals binding (Ni or Zn) and a conserved GKGGVGKS motif in the phosphate binding loop (P-loop), present in the N-terminal domain. Different crystal structures of ChCooC1 are available, while there is no structure of RrCooC.

According to the ChCooC1 structure obtained in the presence of Zn, CooC is a monomer and dimerize upon addition of Zn and ADP (Figure 33A) to form a presumably active homodimer. The metal is covalently bound to the interface of two monomers by two cysteine residues from each monomer (Cys112 and Cys114) (Figure 33B). The structures show that ADP binding induces conformational changes in the metal site and the distances between Zn and the thiolates of Cys114 become shorter (from 2.5 to 2.2 Å) (J.

H. [START_REF] Jeoung | Crystal structure of the ATP-dependent maturation factor of Ni,Fe-containing carbon monoxide dehydrogenases[END_REF]. For RrCooC, the available information comes from its purification from R. rubrum cells.

Once purified, the protein is a homodimer, containing less than 0.1 atoms of Ni 2+ or Zn 2+

and can catalyze the hydrolysis of ATP or GTP [START_REF] Jeon | Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum *[END_REF]. The RrCooC-K13Q mutant presents a mutation in the P-loop and was inactive for both the ATP hydrolysis in vitro and nickel insertion in the Fe-CODH in vivo [START_REF] Jeon | Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum *[END_REF]. The in vitro nickel activation of Fe-CODH in cell extracts (in the presence of CooCTJ), was enhanced by the addition of ATP and Ni salt, while GTP and Ni salt caused a loss of activity, suggesting that ATP is the physiological cofactor in RrCooC [START_REF] Jeon | Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum *[END_REF]. Recent phylogenetic analysis revealed the existence of at least 111 CooT homologues in anaerobic bacteria and archaea, with a strictly conserved cysteine in position 2 [START_REF] Timm | The CO dehydrogenase accessory protein CooT is a novel nickel-binding protein[END_REF]. Among the cooT-containing genomes, 83 contain also CooC and CODH, strongly suggesting its role in CODH maturation, which is probably more widespread than previously expected (Alfano et al., 2018). ChCooT, is preserved in 70.3 % of all sequences, the "Cys2-Cys4" motif, (not characterized to date) present in 3.3 % of all sequences and observed only in archaeal CooT, and the remaining sequences displays a "Cys2" motif, present in RrCooT [START_REF] Alfano | A Solvent-Exposed Cysteine Forms a Peculiar NiII-Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum[END_REF]. In the genomes, the cooT gene can be present alone (C. hydrogenoformans), in duo with

CooC and sometimes in several copies as observed in Thermococcus sp. AM4 (Figure 31)

or in trio with cooJ and CooC, as observed in Thermococcus onnurineus or R. rubrum (Alfano et al., 2018). The coordination of Ni(II) to CooT depends on the nature of the nickel binding motif. Site-directed mutagenesis and biophysical studies showed that both Cys2 and His55 are involved in Ni(II) binding to ChCooT. In RrCooT, Cys2 provides the four Ni ligands (Figure 36A). This bisamidate/bisthiolate Ni(II) coordination via a single cysteine, is unique in biology and raises the question of its role in CODH activation.

Figure 36 Ni(II) binding motifs: (A) RrCooT where the Ni(II) is probably coordinated by two groups thiolate and amidate of both residues of Cys2 and (B)

ChCooT where the Ni(II) is probably coordinated by two Cys2 thiolates and two nitrogen from His55 residues. (Alfano et al., 2018) CooJ from R. rubrum is a 12.5 kDa metallochaperone that contains a His-rich region (HRR) in the C-terminal, formed by 16 His and 2 Cys. CooJ was first purified directly from R. rubrum cells [START_REF] Watt | The Identification, Purification, and Characterization of CooJ[END_REF]. Interestingly, during the purification CooJ was eluted with CODH and other proteins strongly suggesting the formation of a multiprotein complex between CODH, CooJ and other partners. Recently, CooJ was shown to form a homodimer containing a central coil coiled domain flanked by two independent and highly flexible histidine tails (Figure 38). The His tails possess four metal binding sites with a KD of 1.6 µM, similar to that was previously observed [START_REF] Alfano | A Solvent-Exposed Cysteine Forms a Peculiar NiII-Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum[END_REF]. Furthermore, the dimer possesses an additional Ni binding site at the interface of the dimer, which binds Ni(II) with an affinity of 0.38 µM and is defined as a high-affinity site (HAS) [START_REF] Alfano | A Solvent-Exposed Cysteine Forms a Peculiar NiII-Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum[END_REF].

This study also reveals the presence of 46 bacterial homologues, all of which possess two spatially separated nickel binding motifs: a variable C-terminal HRR and a strictly is still able to activate CODH in vivo, suggesting that its tails are dispensable for CODH maturation [START_REF] Kerby | In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: Molecular and physiological characterization of cooCTJ[END_REF]. The role of Ni-binding HRR in proteins is generally proposed to be related to nickel storage and/or detoxification. HRR are also present in other Ni-chaperones with variable location in the protein sequence (C-or N-terminal), such as UreE (N-terminal) and UreG (C-terminal) involved on the maturation of urease or HypB (N-terminal) and SlyD (C-terminal) and HypB (internal position) for the maturation of hydrogenase [START_REF] Alfano | A Solvent-Exposed Cysteine Forms a Peculiar NiII-Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum[END_REF]. The X-ray structure of a truncated RrCooJ, without its His tails, is available (Figure 37A). The structure shows that the HAS is exposed to the solvent, which is compatible with a rapid metal transfer to the physiological partner(s) [START_REF] Alfano | A Solvent-Exposed Cysteine Forms a Peculiar NiII-Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum[END_REF]. The Ni(II) ion is coordinated by four histidine residues, two from each monomer. In the crystal structure two additional Ni ligands from a glutamate present in a close dimer completes an octahedral coordination geometry (Figure 37B), where in a solventexposed Ni(II) binding site, water molecules can easily replace the glutamate residue in solution [START_REF] Alfano | A Solvent-Exposed Cysteine Forms a Peculiar NiII-Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum[END_REF]. To date, the maturation mechanism of CODH remains unclear. Two models have been proposed, implying CooC and CooJ (Figure 38) [START_REF] Jeon | Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum *[END_REF]. In the first one, CooJ binds Ni(II) at first and transfer it to apo-CooC which acts as an insertase to deliver the Ni to the CODH active site. In the second model, CooC uses ATP hydrolysis to fold the nickel center and CooJ would be in charge of delivering nickel to the CODH active site. The role of CooT in these models has not been taken into account and it has yet to be clarified [START_REF] Kerby | In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: Molecular and physiological characterization of cooCTJ[END_REF].

To conclude, the maturation of CODH appears to function differently among microorganism, based on the presence and genomic context of CooC, CooT and CooJ.

The ability to produce recombinant CODH from different biological sources in various heterologous systems sheds light on these mechanisms.

Figure 38 Two proposed model for CooC-CooJ mediated Ni insertion into [Fe]-CODH: (A) CooJ binds the free nickel in the cell and transfer it to the apo-CooC. The ATP hydrolysis allow the transfer of the Nickel from CooJ to the [Fe]-CODH to make the [NiFe]-CODH. (B) CooC use the ATP hydrolysis to fold its proper nickel centre which allow its transfer to CooJ that finally insert the Ni into the [Fe]-CODH to make the [NiFe]-CODH.

The possibility of heterologously produce CODH in the easy-to-grow bacterium E. coli with plasmids containing both the target enzyme and the accessory proteins has opened up a new way in the study of CODH, known to be difficult to purify from natural bacteria (H. [START_REF] Inoue | Over-expression of carbon monoxide dehydrogenase-I with an accessory protein co-expression: A key enzyme for carbon dioxide reduction[END_REF]. For example, the growth of R. rubrum in order to produce CODH requires anaerobic conditions and management of CO, a toxic gas, which also results in a low yield of pure enzymes, 0.3-3 mg L -1 (starting from 107-135 grams of crude cells) [START_REF] Bonam | Purification and characterization of carbon monoxide dehydrogenase, a nickel, zinc, iron-sulfur protein, from Rhodospirillum rubrum[END_REF]) [START_REF] Bonam | Nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: in vivo and in vitro activation by exogenous nickel[END_REF]. Furthermore, when the industrial applications are the final goal, it is essential to develop a stable and reproducible expression and purification system for the large-scale production of enzymes. However, it is important to consider that in the case of metalloproteins, the establishment of an efficient overexpression system is not an easy challenge, due to the complexity of the maturation processes necessary to build and insert their metallic active centers [START_REF] Windsor | Expression of recombinant matrix metalloproteinases in Escherichia coli[END_REF]. To date, several groups have succeeded in the recombinant production of CODHs in E. coli or other host bacteria.

First of all, in 2007 Jeoung and Dobbek expressed and purified ChCODH-II, which has no associated chaperone in the native operon (J.-H. Jeoung & Dobbek, 2007). His-tagged

RecChCODH-II was produced anaerobically in E. coli under reducing conditions with a nickel supply in the culture medium (200 µM NiCl2). Thanks to the His tag, the enzyme was purified in one step and showed a specific CO oxidation activity of 12 229 U•mg -1 , which is close to that of the native enzyme (14 200 U•mg -1 ). The activity was assayed in the same conditions as the wild type (at 70 °C in buffer at pH 8.0).

In [START_REF] Inoue | Over-expression of carbon monoxide dehydrogenase-I with an accessory protein co-expression: A key enzyme for carbon dioxide reduction[END_REF]. However, these results would suggest that additional partners seem to be required for the production of fully mature enzyme, which may not be in the same operon as ChCODH-I and ChCooC3.

ChCODH-I (CooC3 present in the operon)

Wild Type 1.42 18,900 [START_REF] Svetlitchnyi | Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxideutilizing eubacterium Carboxydothermus hydrogenoformans[END_REF] Rec-CODH 0.2 2,270 (T. [START_REF] Inoue | Over-expression of carbon monoxide dehydrogenase-I with an accessory protein co-expression: A key enzyme for carbon dioxide reduction[END_REF] Rec-CODH+CooC3 0.6 8,060

A few years later, the recombinant DvCODH was successfully produce, not in E. coli but in Desulfovibrio fructosovorans [START_REF] Hadj-Saïd | The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris[END_REF]. D. fructosovorans cells were grown anaerobically in a nickel supplemented medium. To date, the wild type enzyme

has not yet been purified. The DvCODH operon harbors the accessory protein CooC. Pure

RecDvCODH displays a very low specific activity (<5 U•mg -1 ) and its co-expression with

CooC has a positive effect on the specific activity (160 U•mg -1 ). Although this value is still far from those obtained with described CODH, these results confirm the importance of CooC when present in the operon. RecDvCODH CooC can be further activated by incubation with NiCl2 under reductive conditions, leading to a specific activity of 1 660

U•mg -1 .
Although the specific activity of the native enzyme is not known, it is possible to hypothesize that some accessory proteins would be missing to heterologously produce a fully active DvCODH, considering the improvement of the activity by in vitro incubation with Ni salts. Recently the same group has performed the production of the recombinant CODH-I and -II from Thermococcus sp. AM4 in Desulfovibrio fructosovorans using the same protocol [START_REF] Benvenuti | The two CO-dehydrogenases of Thermococcus sp. AM4[END_REF]. Again, for TcCODHs, the specific activities of wild type enzymes

are not yet known. The genome of Thermococcus sp. AM4 possesses three cooC genes:

cooC1 inside the CODH-I operon, cooC2 inside the CODH-II operon and an additional cooC3 inside an operon not related to CODH. RecTcCODH-I and -II were co-expressed with the three different cooC genes. As observed with RecDvCODH, the pure RecTcCODH-I and -II showed a higher specific activity and Ni content when they are coproduced with CooC, except in the case of CooC3 (Table 9). In addition, TcCODH-I reaches a better activity when it has been co-expressed with CooC2 (twice compared to the co-expression with CooC1) whose gene is present in the same operon as CODH-II, suggesting that CooC can be interchangeable for CODH activation (Figure 39). Another question arises on the role of CooC3, which is not present in any TcCODHs operon (Figure 39) and its co-expression with both TcCODHs does not have any impact on the enzyme activity. Furthermore, the low activities obtained in all combinations of CODH and CooC genes strongly suggest that, in addition to CooC, other metallochaperones are required for the C-cluster biosynthesis. In fact, in the TcCODH-II 39). Their presence strongly suggests their implication in the maturation pathway of the enzyme. [NiFe]-CODHs have been studied in depth by various approaches, such as X-ray crystallography, spectroscopy and electrochemistry, allowing the postulation of a common catalytic mechanism. First of all, several CODH were co-crystallized with substrates/products (CO, CO2 and H2O), analogues of substrates and products as well as inhibitors (cyanide, cyanate, n-butyl isocyanate). The 2.0 Å resolution structure of MbCODH (W. [START_REF] Gong | Structure of the α2ε2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex[END_REF] shows that CO is bound to Ni and a water molecule is bound to the Feu (Figure 40A). These results are supported by the MtCODH/ACS structure at 2.15 Å resolution [START_REF] Kung | Crystallographic snapshots of cyanide-and water-bound C-clusters from bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase[END_REF] which was solved after crystal soaking with cyanide (CN -a CODH inhibitor). CN is bound to Ni and a water molecule is bound to Feu (Figure 40B). In both structures, CO/CN -appears in a bent geometry, confirmed in the ChCODH structure co-crystallized with n-butyl isocyanate (n-BIC) (J. H. [START_REF] Jeoung | n-Butyl isocyanide oxidation at the [NiFe 4S 4OH x] cluster of CO dehydrogenase[END_REF], where n-BIC is bound to Ni and water still bound to Feu (Figure 40C). This bending is due to the steric hindrance of a conserved isoleucine. to create room leading to a more stable linear geometry [START_REF] Ciaccafava | When the inhibitor tells more than the substrate: The cyanide-bound state of a carbon monoxide dehydrogenase[END_REF].

Furthermore, the ChCODH structures in the presence of either CO2 or cyanate (NCO -) (Figure 41BC), were solved at the real atomic resolutions of 1.03 and 1.06 Å, respectively. In both structures, CO2/NCO -carbon is bound to Ni and oxygen to Feu. In the case of CO2, the oxygen bound to Feu comes from a water molecule. 1990) [START_REF] Krzycki | Paramagnetic centers of carbon monoxide dehydrogenase from aceticlastic Methanosarcina barkeri[END_REF][START_REF] Hadj-Saïd | The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris[END_REF]. a [START_REF] Spangler | Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum[END_REF], b [START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF], c [START_REF] Lindahl | CO Dehydrogenase from Clostridium Thermoaceticum[END_REF], d [START_REF] Krzycki | Paramagnetic centers of carbon monoxide dehydrogenase from aceticlastic Methanosarcina barkeri[END_REF]), e [START_REF] Hadj-Saïd | The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris[END_REF] The typical EPR spectra of RrCODH at different redox potentials are shown in Figure 43A ( [START_REF] Spangler | Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum[END_REF]. The [4Fe4S] clusters also show a characteristic UV-visible absorbance between 350 to 480 nm, as shown in Figure 43B [START_REF] Ensign | Nickel is required for the transfer of electrons from carbon monoxide to the iron-sulfur center(s) of carbon monoxide dehydrogenase from Rhodospirillum rubrum[END_REF]. [START_REF] Ensign | Nickel is required for the transfer of electrons from carbon monoxide to the iron-sulfur center(s) of carbon monoxide dehydrogenase from Rhodospirillum rubrum[END_REF].

Different molecules can act as CODH inhibitors. The attention will be focused on the inhibitors that have contributed to the postulation of the catalytic mechanisms. Sulfide (S 2-, HS -or H2S) has been proposed to act both as inhibitors and as activators.

Sulfide inhibits the oxidation of CO, but not the reduction of CO2, they bind the Cox state by inhibiting catalytic activity at potentials from -50 to -250 mV [START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF].

Cyanide, an analogue of CO, is a reversible inhibitor of CO oxidation, which binds the Cred1 state. But CN -does not interact with the Cred2 state and does not inhibit the reduction of CO2. As shown above, CN -has different binding modes, so CN -can bind Ni in a bent mode like CO and acts as a competitive inhibitor. A subsequent slow rearrangement step can induce the displacement of the water molecule bound to Feu and leave room for CN -relaxation allowing a linear binding mode, in addition to producing a more stable cyanide adduct (Figure 44) [START_REF] Kung | A role for nickel-iron cofactors in biological carbon monoxide and carbon dioxide utilization[END_REF].

Isocyanides (e.g., n-BIC) can act both as a substrate and as an inhibitor of CO oxidation.

CODH catalyzes the oxidation of n-BIC to n-BICt 10 5 times slower than CO. In this way, n-BIC acts as a competitive Inhibitor of CO [START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF].

Cyanate, an analogue of CO2, binds the Cred2 state and inhibits the reduction of CO2.

Conversely it does not interact with the Cred1 state and does not inhibit the oxidation of CO. The binding of NCO -takes several seconds with millimolar concentrations and inhibition occurs at more positive potentials of -400 mV [START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF].

All together this information led to the postulation of the following catalytic mechanism of CODH (Figure 44

Product releasing:

CO2 is released, and the cluster is further reduced by two electrons to reach the Cred2 state. A new H2O binds the Fe(II) ion. Concerning the gaseous substrate, as shown above, MtCODH/ACS displays gas channels for CO/CO2 transfers from the CODH subunit to the ACS. Furthermore, the ChCODH structure co-crystallized with n-BIC shows a second n-BIC molecule bound to the enzyme which could represent the gateway from the solvent to the C-cluster (Figure 45A). A polar channel is needed to transport the protons to and from the active site. All CODH structures contain a channel of water molecules coated with several ionizable histidine and a lysine residues, the latter being very close to the Feu-H2O (N-Lys at 2.3 Å from O of Feu-H2O) (Figure 45B). Through this gateway, protons can be transferred to C-cluster for CO2 reduction or to the solvent during CO oxidation. In addition, there is also a gateway for the H2O substrate, composed of conserved structural water molecules (Figure 45B). Monofunctional CODHs can catalyze CO/CO2 interconversion, but there is a significant difference in terms of turnover frequency between CO oxidation and CO2 reduction (Table 11). Furthermore, the efficiency of CO oxidation varies considerably from one enzyme to another (Table 11).

Regeneration (Cred2

CO oxidation is characterized either by electrochemistry, usually with an enzyme immobilized on an electrode, or in solution following the CO-dependent reduction of oxidized chemical acceptors. CODHs catalyze the CO-dependent reduction of various electron acceptors, such as methyl-and benzyl-viologen, phenazine metazose, methylene blue, etc. Among them, methyl-viologen (MV) is the most used for the determination of the specific activity of CODHs, by following its reduction over time at 604 nm (ε = 13.6 mM -1 • cm -1 ), from colorless to violet.

The reduction of CO2 is both characterized by electrochemistry and in solution. In solution, the reduction of CO2 to CO by CODH is measured by monitoring the spectrophotometric absorbance changes upon the binding of CO to reduced hemoglobin (shift of the Soret band from 433 to 419 nm, ε = 185 mM -1 • cm -1 ), to form carbonyl-hemoglobin, by using NaHCO3 as a CO2 source. CODHs specific activities are expressed in U • mg -1 (µmol of oxidized CO or reduced CO2 • min -1 • mg -1 ).

CO2 + 2e -+ 2H + + Hb → H2O + Hb-CO

The studies on the redox dependence of the reduction of CO2 by RrCODH confirm that CO2 is not able to bind the Cred1 state but is able to bind the Cred2 state because the activity increase of 2000 times when the potential is less than -500 mV [START_REF] Heo | Redox-dependent CO2 reduction activity of CO dehydrogenase from Rhodospirillum rubrum[END_REF]. Ch I 8 70 18900 (Svetlitchnyi et al., 2001) Ch II 8 70 14200 (Svetlitchnyi et al., 2001) Tc II 6 37 70 [START_REF] Benvenuti | The two CO-dehydrogenases of Thermococcus sp. AM4[END_REF] 6 37 0.3 [START_REF] Benvenuti | The two CO-dehydrogenases of Thermococcus sp. AM4[END_REF] To date, several electrochemical studies on CODH activities have been conducted.

Furthermore, the kinetics properties of the substrates (CO and CO2) and of the inhibitors (CN -and NCO -) were elucidated by electrochemistry. But these results will be presented in the following sections, dedicated to the advantages of bio-electrochemistry for the study of redox metalloenzymes such as CODH. ( [START_REF] Holzinger | Nanomaterials for biosensing applications: a review[END_REF].

In electrochemistry, one of the natural co-substrate/s, cofactor/s or redox partner/s is replaced by an artificial molecule (mediator), or the natural redox partner is electrochemically regenerated by an artificial molecule. This type of electron transfer mechanism is called mediated electron transfer (MET) (Figure 47A). Alternatively, if the cofactor is electrochemically regenerated at the electrode, this type of electron transfer mechanism is called direct electron transfer (DET) (Figure 47B). The transfer of an electron between two molecules in solution should be thermodynamically favorable and the difference in energy levels is the driving force of the reaction [START_REF] Rountree | A Practical Beginner ' s Guide to Cyclic Voltammetry[END_REF].

The driving force is expressed in terms of the variation of the Gibbs free energy of the reaction, the distance between the redox centers and the reorganization energy [START_REF] Vidakovic-Koch | Electron Transfer Between Enzymes and Electrodes[END_REF]. DET was first demonstrated about 40 years ago [START_REF] Yaropolov | 192 -Electrochemical Properties of Peroxidase[END_REF]) [START_REF] Tarasevich | Electrocatalysis of a cathodic oxygen reduction by laccase[END_REF]) [START_REF] Armstrong | Direct electrochemistry of redox proteins[END_REF]. [START_REF] Miller | Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar-Driven Reduction of Carbon Dioxide[END_REF]. DET has also been reported for enzymes in which the active site is buried within the protein, such as [NiFe]-Hydrogenases [START_REF] Léger | Effect of a Dispersion of Interfacial Electron Transfer Rates on Steady State Catalytic Electron Transport in [NiFe]-hydrogenase and Other Enzymes[END_REF]. This is possible thanks to the presence of FeS clusters which reduces the distance between the active center and the surface of the electrode.

In fact, the electron transfer rate is inversely proportional to the distance between the enzymatic active site and the electrode surface [START_REF] Jeuken | Conformational reorganisation in interfacial protein electron transfer[END_REF]. In support of this it has been shown that DET is observable for distances less than 25 Å [START_REF] Agbo | Modeling Dioxygen Reduction at Multicopper Oxidase Cathodes[END_REF].

It is therefore evident that the efficiency of DET strongly depends on the orientation of the enzyme on the surface of the electrode and on the electronic and chemical properties of the electrode. The advantage of DET is that it allows the bioelectrocatalysis at the redox potential of the enzyme, which is generally close to the redox potential of the substrate/product involved in the reaction. However, considering that in the enzyme only a small area allows DET, the orientation of the enzyme on the electrode surface is essential to obtain an efficient DET. In fact, in some cases the chemical properties of the protein can favor or disadvantage the DET of the immobilized enzyme and the addition of a redox mediator can improve the quantity of electroactive enzyme and then the catalytic current (Figure 48) [START_REF] Ciaccafava | Electrochemistry , AFM , and PM-IRRA Spectroscopy of Immobilized Hydrogenase : Role of a Hydrophobic Helix in Enzyme Orientation for Efficient H 2 Oxidation ** Angewandte[END_REF]. The mediator can be a small molecule (as represented in Figure 48) but also an electroactive polymer or gel in which the enzyme is immobilized. However, in the presence of the mediator, the potential of electrocatalysis is shifted from that of the enzyme to the mediator redox potential. Hence, it is clear that to obtain an efficient DET it is essential to optimize the immobilization of the enzyme on the electrode surface.

To date, various enzymatic immobilization techniques have been exploited in order to immobilize and electrically wire enzymes to electrodes, such as: physical adsorption, cross-linking, entrapment or encapsulation, covalent and supramolecular assembly. It is important to consider that immobilization should not affect the stability of the enzyme, such as alteration of the structure or partial unfolding, or modify its catalytic properties.

Furthermore, the transport kinetics of the substrate/s and product/s can also be disturbed by immobilization.

Physical adsorption is the simplest immobilization technique, the enzyme is adsorbed on the surface via hydrophobic, electrostatic or Van de Waals interactions (Figure 48A).

The advantage is simplicity and the absence of any chemical process that might be detrimental to enzyme activity or accessibility of the substrate. On the other hand, due to the soft interactions, it is possible to observe the desorption of the enzyme, especially under experimental conditions that reduce these interactions.

Cross-linking consists in the use of a bifunctional reagent such as glutaraldehyde, which is the most used reticulant [START_REF] Migneault | Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking[END_REF]) [START_REF] Sheldon | Enzyme immobilisation in biocatalysis: Why, what and how[END_REF], which reacts with amines of proteins inducing their reticulation and precipitation on the electrode surface (Figure 48B). Compared to physical adsorption, stability over time is improved, but the cross-linking can induce partial unfolding or a loss of coordination of the cofactors, thereby reducing catalytic performances. More accurate immobilization can be achieved by exploiting bioinspired interactions of molecules, such as between saccharides and lectins or specific chemical groups with specific protein pocket. Particular attention must be paid to the functionalization of the electrode and/or the enzyme in order to enable affinity interactions. The electrode surface is functionalized with compounds such as polymers, diazonium salts or by forming a self-assembly monolayer (SAM) of enzyme [START_REF] Samanta | Immobilization of bio-macromolecules on selfassembled monolayers: methods and sensor applications[END_REF]) [START_REF] Cosnier | Recent Advances in Biological Sensors Based on Electrogenerated Polymers: A Review[END_REF]. For example, a SAM can be generated by exploiting the biotin/streptavidin chemistry. First the electrode and proteins are functionalized with biotin, then the streptavidin is added to induce the specific interactions (Figure 50A). Another method consists in the use of ferrocene or adamantane cyclodextrin derivates (Figure 50B) [START_REF] Villalonga | Supramolecular chemistry of cyclodextrins in enzyme technology[END_REF]) [START_REF] Dubacheva | Electrochemically controlled adsorption of Fcfunctionalized polymers on beta-CD-modified self-assembled monolayers[END_REF]) (Cosnier & Holzinger, 2011). A third method is the well-known nitrilotriacetic acid (NTA)-Cu chemistry to provide affinity interactions with histidine tagged proteins (Figure 50C) [START_REF] Haddour | Electrogeneration of a poly(pyrrole)-NTA chelator film for a reversible oriented immobilization of histidine-tagged proteins[END_REF]) [START_REF] Balland | Oriented immobilization of a fully active monolayer of histidine-tagged recombinant laccase on modified gold electrodes[END_REF]. Other examples are based on electrostatic and hydrophobic interactions (Holzinger et al., 2014). The advantage of these techniques is to provide a specific orientation of the molecules to the electrode surface, but the modification of the proteins can also alter their structure and therefore their catalytic properties.

Finally, a covalent immobilization can be used, which required a preliminary functionalization of the electrode surface in order to provide the chemical groups for the covalent immobilization of proteins. Generally, covalent bonding involves nucleophilic proteins residues such as lysine and cysteine which react with NHS esters or aldehydes, maleimide and epoxides respectively (Figure 51A) [START_REF] Wong | Selective Covalent Protein Immobilization : Strategies and Applications[END_REF].

These techniques provide strong immobilization but do not control orientation, resulting in a mixture of DET-electroactive species. To better control the orientation and maintain the covalently binding of the protein can alternatively be modified with azide to exploit the click chemistry or the Diels-Alder cycloadditions (Figure 51B) [START_REF] Gentil | Efficiency of Site-Specific Clicked Laccase-Carbon Nanotubes Biocathodes towards O2 Reduction[END_REF]) [START_REF] Gentil | Clicked Bifunctional Dendrimeric and Cyclopeptidic Addressable Redox Scaffolds for the Functionalization of Carbon Nanotubes with Redox Molecules and Enzymes[END_REF]. Thus, each strategy of enzyme immobilization possesses advantage and drawbacks. For example, the conductive polymers increase the quantity of entrapped enzyme (tridimensional matrix) but does not control its orientation, resulting in portion of nonelectroactive enzyme. This is counterbalanced by the modification of the polymer backbone with redox mediators, enabling electron transport throughout the film. On the contrary, the SAM and covalent immobilization favor a specific orientation.

However, the amount of enzyme is limited by the bidimensional electrode surface. Then, an ideal electrode should provide a tridimensional conductive surface and allow the orientated immobilization without interfering with in the catalysis and protein stability.

The next section will be focused on the use of carbon nanotubes (CNT)-based electrode for enzymatic immobilization.

Carbon-based nanomaterials such as graphene, carbon black and CNT, are the most suitable materials for enzymatic immobilization for several reasons: they are cheaper than other supports (e.g., gold), afford a good conductivity, a high surface volume ratio and the possibility to easily modify the surface structure. Among these carbon-based nanomaterials, CNTs are particularly attractive because they offer the ideal characteristics to balance the key factors that determine the efficiency of biocatalysts.

CNT-based sensors generally have higher sensitivities, lower detection limits and faster electron transfer kinetics than traditional carbon electrodes (W. [START_REF] Feng | Enzymes immobilized on carbon nanotubes[END_REF]. The performance of the CNT-enzyme based device is influenced by a combination of nanotube chemistry and the immobilization method. It is therefore essential to find optimal methods for CNT functionalization for enzyme immobilization and electric wiring.

CNT are seamless cylinders made of carbon lattice (graphene) with open or closed ends, with diameters typically measured in nanometers. CNT can be made up of a closed wrapped-up graphene layer, called SWCNT discovered independently in 1993 by two research groups (Figure 52) [START_REF] Iijima | Single-shell carbon nanotubes of 1-nm diameter[END_REF]) [START_REF] Bethune | Cobalt-catalysed growth of carbon nanotubes with singleatomic-layer walls[END_REF]. On the other hand CNT can consist of nested SWCNT (at least two SWCNT) called MWCNT, discovered in 1991 (Figure 52) [START_REF] Iijima | Helical microtubules of graphitic carbon[END_REF]. The diameters of SWCNT and MWCNT are generally from 0.4 to 3 nm and 1.4 to 100 nm, respectively, although the MWCNT diameters can exceed the nanoscale (> 100 nm) [START_REF] Baughman | Carbon nanotubes--the route toward applications[END_REF]. The lengths of the CNT range from less than 100 nm to several centimeters, covering thus molecular and macroscopic scales [START_REF] Baughman | Carbon nanotubes--the route toward applications[END_REF]. The CNT have all the sp 2 carbon atoms bound in a hexagonal lattice except at the extremities. The composition of the lattice can also present defects introducing pentagons, heptagons and other imperfections in the sidewalls, which generally change the properties of the CNT [START_REF] De | Carbon nanotubes: Present and future commercial applications[END_REF]. Some carbons are sp 3 which present oxygen-containing functional groups such as alcohols, carbonyl or carboxylic acid.

The CNT show unique properties such as remarkable electrical conductivity. The individual CNT walls can be metallic or semiconductor depending on the spatial orientation of the graphene lattice with respect to the axis of the tube, the latter is called CNT chirality. MWCNT are typically metallic and can carry currents of up to 109 A•cm -2 [START_REF] Wei | Reliability and current carrying capacity of carbon nanotubes[END_REF]. CNT also have exceptional tensile strength: considering only the crosssectional area of the CNT walls. An elastic modulus has been measured that approaches 1 TPa and a tensile strength of 100 GPa for individual MWCNT [START_REF] Peng | Measurements of near-ultimate strength for multiwalled carbon nanotubes and irradiation-induced crosslinking improvements[END_REF]. This resistance is more than 10 times higher than any industrial fiber. Another interesting property is their thermal conductivity. The individual SWCNT shows a thermal conductivity of 3500 W•m -1 •K -1 at room temperature [START_REF] Pop | Thermal Conductance of an Individual Single-Wall Carbon Nanotube above Room Temperature[END_REF], based on the area of the wall, exceeding thus the thermal conductivity of diamond. These properties are expected to be invaluable in many technological areas, such as electronics, optics, composite materials, nanotechnology and other materials science applications [START_REF] De | Carbon nanotubes: Present and future commercial applications[END_REF]. The solubility of CNT in organic and aqueous solvents has been extensively studied [START_REF] O'connell | Carbon Nanotubes Properties and Applications: Properties and Applications[END_REF]. Furthermore, the use of surfactants or the encapsulation into polymers allow the dispersion of CNT. It is important to consider that CNT dispersion should ideally not modify the properties of the CNT (O'Connell, 2006).

Hence, a deposition of CNT film on a surface can be obtained with different strategies:

• The Langmuir-Blodgett technique consists in the dispersion of CNT in aqueous solvents and then in the deposition layer-by-layer (up to 100) on the surface, which allow the control of the film thickness [START_REF] Kim | Langmuir-Blodgett Films of Single-Wall Carbon Nanotubes: Layer-by-layer Deposition and In-plane Orientation of Tubes[END_REF].

• Self-assembly is based on a first surface functionalization with polar (amino or carboxyl) or non-polar (methyl) groups, which drives the self-assembly of the individual CNT (Rao et al., 2003).

• The dip-coating consists in the deposition of a dispersed solution of CNT directly on the surface and subsequently the film is made with high-speed rotation (spincoating) or drying (drop-casting) [START_REF] Saran | Fabrication and Characterization of Thin Films of Single-Walled Carbon Nanotube Bundles on Flexible Plastic Substrates[END_REF].

• Other methods consists in the pre-formation of the CNT film and then in the transfer to the final surface via soluble membranes or an adhesive surface [START_REF] Zhu | Active and Selective Conversion of CO 2 to CO on Ultrathin Au Nanowires[END_REF])(M. [START_REF] Zhang | Strong, transparent, multifunctional, carbon nanotube sheets[END_REF] or by means of a micro-contact printing technique [START_REF] Fuchsberger | Multiwalled Carbon-Nanotube-Functionalized Microelectrode Arrays Fabricated by Microcontact Printing: Platform for Studying Chemical and Electrical Neuronal Signaling[END_REF].

When the goal is the combination of CNT with biological molecules, such as proteins and DNA it is important to consider that CNT can be conveniently functionalized to improve their biocompatibility and biodegradation. Therefore, it is not surprising that the between the sidewalls of the CNT and the aromatic rings of certain amino acids such as phenylalanine [START_REF] Matsuura | Selectivity of water-soluble proteins in single-walled carbon nanotube dispersions[END_REF]. Electrostatic interaction has also been demonstrated in the adsorption of lysozyme on CNT [START_REF] Nepal | pH-sensitive dispersion and debundling of singlewalled carbon nanotubes: lysozyme as a tool[END_REF]. In In 2001, the possibility of immobilizing a protein via amide bond on π-π stacking functionalized CNT was achieved with 1-Pyrenebutanoic acid, succinimidyl ester, involving the nucleophilic substitution of N-hydroxysuccinimide by an amine group of the protein (Figure 53B) (R. J. [START_REF] Chen | Noncovalent Sidewall Functionalization of Single-Walled Carbon Nanotubes for Protein Immobilization[END_REF]. Since this first report of pyrene-derivate molecule to date several other molecules have been described for the development of bioelectrodes for enzyme immobilization and wiring (Table 12). was also used for the oriented immobilization of [NiFeSe]-hydrogenase [START_REF] Gentil | Oriented Immobilization of [NiFeSe] Hydrogenases on Covalently and Noncovalently Functionalized Carbon Nanotubes for H 2 /Air Enzymatic Fuel Cells[END_REF]. Electrochemistry, quartz crystal microbalance with dissipation monitoring and theoretical calculations show that the enzyme was oriented efficiently to allow direct transfer of electrons from the active site of hydrogenase to CNT. the adamantanepyrene was also employed for the oriented immobilization of laccase [START_REF] Lalaoui | Hosting adamantane in the substrate pocket of laccase : direct bioelectrocatalytic reduction of O2 on functionalized carbon nanotubes Hosting adamantane in the substrate pocket of laccase : direct bioelectrocatalytic reduction of O 2 on functionalized car[END_REF]. Like anthraquinone derivatives, the pyrene moiety was also covalently linked to the anthracene groups to produce a molecule capable of functionalizing the CNT [START_REF] Giroud | Anthracene-modified pyrenes immobilized on carbon nanotubes for direct electroreduction of O2 by laccase[END_REF]. These studies showed that anthracene groups are able to orientate the immobilization of the laccase active site near the CNT and allow a rapid direct electron transfer. A pyrene-boronic acid was exploited for the immobilization of glycosylated enzyme, such as the horseradish peroxidase (HRP) via the formation of a covalent boronic ester bond [START_REF] Reuillard | Non-covalent functionalization of carbon nanotubes with boronic acids for the wiring of glycosylated redox enzymes in oxygen-reducing[END_REF]. Another example of pyrene derivates compounds are pyrene-methylammonium and pyrene-carboxylates to introduce negative and positive charges to the surfaces of CNT for the immobilization of hydrogenases or bilirubin oxidases (BOD). [START_REF] Li | Synthesis of water-soluble phosphonate functionalized single-walled carbon nanotubes and their applications in biosensing[END_REF], reported an easy functionalization of SWCNT with naphthalen-1-methylphosphoric acid by π-π stacking, the functionalized CNT showed a negative charge from the phosphonate groups which were used for the immobilization of positively charged myoglobin through electrostatic interaction. This biosensor has shown excellent bio-electrocatalytic activity towards the reduction of hydrogen peroxide with a detection limit between micro and nano-molar scale [START_REF] Li | Synthesis of water-soluble phosphonate functionalized single-walled carbon nanotubes and their applications in biosensing[END_REF].

The π-π stacking functionalization of CNT sidewalls with porphyrins (protoporphyrin IX and Hemin) was exploited to induce the substrate-mimicking immobilization of bilirubin oxidase (Lalaoui, Le Goff, et al., 2015). The MWCNT/BOD bioelectrode achieved a high current density towards a direct oxygen reduction of 4 mA•cm -2 .

As previously presented, protein entrapment in a redox polymer offers three main advantages: stable immobilization over time, electronic wiring of the enzymatic active sites to the electrode and enzymatic compartmentalization in the 3D matrix. This can afford in some cases controllable access of the substrate or inhibitors and protection against inhibition, deactivation or denaturation (Le [START_REF] Goff | Molecular engineering of the bio/nano-interface for enzymatic electrocatalysis in fuel cells[END_REF]. Either polyvinylic or conjugated conductive polymers such as polypyrroles have been extensively studied for enzymatic for biosensors and biofuel cell applications. Pyrrole monomers containing pyrene groups or activated ester groups have been used for the hydrophobic and supramolecular immobilization of Laccase from Trametes versicolor [START_REF] Lalaoui | Efficient direct oxygen reduction by laccases attached and oriented on pyrenefunctionalized polypyrrole/carbon nanotube electrodes[END_REF]. Another example of a polypyrroles polymer that exploits the advantage of using methyl viologen groups has been described for the immobilization Furthermore, these defects can be exploited for a subsequent modification with a molecule that allows the supramolecular immobilization of enzymes. For example, Minteer and collaborators functionalized defect-containing CNT with anthracene groups for the immobilization of laccases (Figure 55A) [START_REF] Meredith | Anthracene-Modified Multi-Walled Carbon Nanotubes as Direct Electron Transfer Scaffolds for Enzymatic Oxygen Reduction[END_REF]. Another example is the exploitation of the CNT defect for functionalization with redox mediators, in order to allow MET (Figure 55B) [START_REF] Callegari | Functionalised single wall carbon nanotubes / polypyrrole composites for the preparation of amperometric glucose biosensors[END_REF] On the other hand, the direct functionalization of CNT sidewalls generally proceeds with addition reactions directly on the carbon sp 2 lattice. However, it has been shown that these addition reactions preferably take place at the edge of the tubes or at the defect sites. Therefore, a pre-oxidation step of the CNT is preferable.

Various addition reactions have been described to date, such as carbene or nitrene additions (Figure 56A). Other most exploited methodologies are functionalization with aryldiazonium salts and the Prato reaction. In 1993, Prato first described the 1,3-dipolar cycloaddition for the functionalization of fullerenes and then for CNT in the 2000s. The reaction is carried out at 130 °C in DMF with an aldehyde derivate and an N-substituted glycine which leads to the formation of substituted pyrrolidine residues. Furthermore, this reaction can be carried out on oxidized or non-oxidized CNTs. Described for the first time in 2001, diazonium functionalization has been widely used due to this high functionalization efficiency with different types of phenyl derivates compounds (Figure 56B). It is generally carried out by electro-grafting of the phenyldiazonium salts on the CNT sidewalls or on the in situ formed phenyldiazonium resulting in a growth of a polyphenylene layer. The diazonium salts can provide hydrophobic functional groups in the para position such as anthraquinone, anthracene, naphthalene or chrysene, as shown for example for the immobilization of laccases [START_REF] Blanford | A stable electrode for highpotential, electrocatalytic O2 reduction based on rational attachment of a blue copper oxidase to a graphite surface[END_REF] [START_REF] Blanford | Efficient electrocatalytic oxygen reduction by the 'blue' copper oxidase{,} laccase{,} directly attached to chemically modified carbons[END_REF]. The covalent modification of CNTs with other diazonium salts for the immobilization of redox enzymes have been extensively described. Several hydrogenases were immobilized on CNT functionalized with diazonium salts which provided positive, negative or hydrophobic charges (Alonso-Lomillo et al., 2007) [START_REF] Gentil | Oriented Immobilization of [NiFeSe] Hydrogenases on Covalently and Noncovalently Functionalized Carbon Nanotubes for H 2 /Air Enzymatic Fuel Cells[END_REF].

In the next section attention will be focused on the possibility of integrating these nanobio materials in functional devices such as gas diffusion electrodes and biofuel cells.

When the goal is to develop functional devices for biotechnological applications, such as CO2RR for the production of chemicals, the choice of electrode material has a major impact on the performance of the bioelectrochemical systems. The advantages and disadvantages of biological catalysts and functionalized CNT were discussed above.

However, in all the present examples, these nanobio materials were applied in twophase systems: the solid phase (electrode supported with CNT-biocatalysts) and the liquid phase (electrolyte, substrate(s) and mediator(s)). In this respect, in recent years, gas diffusion electrodes (GDEs) have proved to be very attractive electrodes for bioelectrochemical purposes (Figure 57). GDEs avoid major drawbacks when the catalyzed reaction involves gaseous substrates, such as poor gas solubility, mass transport, acidification or basification of electrolytes, and others. GDEs are porous electrodes that have a three-phase boundary: the solid one supports the catalyst and the electrochemical reaction and is located at the interface between the gas and liquid phases. GDEs are typically implemented into conventional fuel cells including biofuel cells (Figure 57). The latter are bioelectrochemical devices where a chemical reaction takes place and electrical energy is generated from the conversion of chemical energy (Galvanic Cell, ΔG < 0) or vice versa (Electrolysis Cell, ΔG > 0). Generally, a fuel cell consists of an electrolyte chamber in contact with the cathode and the anode. On the anodic side, the oxidation of the fuel is achieved (usually hydrogen from an incoming reservoir is decomposed into protons and electrons) while on the cathodic side the reduction of the oxidant is performed (reduction of O2 to H2O) (Le [START_REF] Goff | Molecular engineering of the bio/nano-interface for enzymatic electrocatalysis in fuel cells[END_REF]. Since the first developments of fuel cells (19 th century) it has been observed that the main limitation is the need for a significant area between gas, electrolyte and electrode [START_REF] Segura | Fuel cells : History and updating . A walk along two centuries[END_REF].

Figure 57 Overview of the different bioelectrochemical application fields for gas diffusion electrodes (GDE).

The most typical application method of GDEs is described for the different fields, respectively. GFC and HCS are for Glucose and Hydrogen Fuel Cells, respectively. MCO is for Multi Copper Oxidases. (Modified from [START_REF] Horst | Application of Gas Diffusion Electrodes in Bioelectrochemical Syntheses and Energy Conversion[END_REF].

This limitation has aroused great interest in GDEs and in their implementation in fuel cells, at the interface between the electrolyte and the gas phase.

The implementation of GDEs in fuel cells occurs in two configurations: a component of the gas phase intervenes in the electrochemical reaction or the desired gaseous compound is produced at the electrode. In the first case, the cathodic GDE carries out the oxygen reduction reaction (ORR), to get around the problem of the low solubility of molecular oxygen in aqueous electrolyte (O2 concentration is about 250 µM at room temperature and atmospheric pressure in pure water). ORR in aqueous solutions occurs mainly through two pathways: the direct 4-electrons reduction from O2 to H2O and the 2-electrons reduction from O2 to H2O2 [START_REF] Horst | Application of Gas Diffusion Electrodes in Bioelectrochemical Syntheses and Energy Conversion[END_REF]. In non-aqueous aprotic solvents and/or alkaline solutions, a 1-electron reduction pathway from O2 to O2 -can also occur [START_REF] Horst | Application of Gas Diffusion Electrodes in Bioelectrochemical Syntheses and Energy Conversion[END_REF]. The second situation is when the anodic GDE performs the hydrogen evolution reaction (HER), the electrons from the cathode are used to reduce the protons to form hydrogen, which diffuses through the GDE to a collector. A standard biofuel cell (Figure 58) contains electrodes where: at the anode a substrate (e.g., glucose) is oxidized by the biocatalysts (enzymes or microbial) to generate electrons and protons. The electrons pass through an outside circuit to the cathode, while the protons pass through an ionic-exchange membrane to reach the cathode where the oxidant such as O2 is reduced to H2O. During this redox process, the generated electric current works to continuously operate electrical devices when it is a closed circuit. In biofuel cells biochemical transformations can be performed by either whole cells or enzymes and are called microbial or enzymatic fuel cells, respectively.

Figure 58 Schematic representations of (A) Microbial fuel cell (MFC)-microorganisms oxidize organic substrates (S), produced electrons reduce the anode and are transferred via an external circuit to the cathode. At the cathode ORR takes place, in this case, 4-electrons reduction from O2 to H2O. No input in form of electricity is needed but energy is harvested as electrical energy. (B) Enzymatic fuel cell (EFC)-enzymes oxidize organic substrates (S) at the anode and transfer electrons to the electrode. At the cathode, oxidases catalyze the ORR to water. Thus, biochemical energy is converted to electrical energy. (C) Microbial electrolysis cell (MEC)-microorganisms transfer electrons to the anode and with an energy supply HER takes place at the microbial-based cathode. (D) Electroenzymatic synthesis (EES)-

oxygen is reduced at the cathode to form hydrogen peroxide. This substance is used as co-substrate for an enzymatic oxidation or halogenation (anodic reaction not shown). (Adapted from [START_REF] Horst | Application of Gas Diffusion Electrodes in Bioelectrochemical Syntheses and Energy Conversion[END_REF] Microbial fuel cells (MFCs) exploit the metabolism of microbes to oxidize organic matter (anode) and transfer electrons from these reactions to the cathode to carry out a reduction reaction (ORR) and generate current (Figure 58A). The first use of GDE in MFC was reported in 1989, where an oxygen GDE was successfully used as a cathode for ORR with E. coli as a catalyst for the oxidation of organic matter and 2-hydroxy-1,4naphthoquinone as a redox mediator, in the anodic compartment [START_REF] Sell | Use of an oxygen gas diffusion cathode at threedimensional packed bed anode in a bioelectrochemical fuel cell[END_REF].

The GDE cathode reached a maximum current density of 1.3 mA•cm -2 . Microorganisms transfer electrons to the anode using several methods, including direct contact of outer membrane cytochromes on the cell surface or on conductive extensions (such as pili which can achieve long-distance electron transfer), through self-produced mediators (such as flavins) which can carry electrons between the cell and the anode [START_REF] Logan | Electroactive microorganisms in bioelectrochemical systems[END_REF]. As an alternative to microorganisms as catalysts for biofuel cells, isolated enzymes can also act as electrochemical catalysts in enzymatic fuel cells (EFCs). EFCs use redox enzymes for the oxidation of organic compounds at the anode and redox enzymes for ORR at the cathode to generate electricity (Figure 58B). Apart from applications of EFCs in harvesting energy from oxygen present in extracellular fluids (blood and interstitial fluid), GDEs are often used as cathodes in EFCs based on enzymes capable of reducing oxygen, such as laccases or bilirubin oxidase (multicopper oxidase or MCO) [START_REF] Osman | Recent progress and continuing challenges in bio-fuel cells. Part I: Enzymatic cells[END_REF]. The most widely studied EFCs are glucose (GFCs) and hydrogen (HFCs) EFCs. GFCs are based on MCO at the cathode for ORR and on glucose oxidizing enzymes at the anode. GFCs for the development of micro-biosensors have been integrated into living organisms such as rats, rabbits, snails, insects, clams and lobsters (Le [START_REF] Goff | Molecular engineering of the bio/nano-interface for enzymatic electrocatalysis in fuel cells[END_REF]. However, in vivo stability is the main bottleneck of GFCs, at they have been reported to be stable for one week, while operating for over one year in vitro (Le [START_REF] Goff | Molecular engineering of the bio/nano-interface for enzymatic electrocatalysis in fuel cells[END_REF]. On the other hand, HFCs use hydrogen oxidizing enzymes, such as hydrogenases, at the anode and MCO for the ORR at the cathode.

Since the efficiency of electrochemical processes is directly proportional to the electrochemically active area, CNT with their inherently high surface-to-volume ratio have been integrated into GDEs. For example, in 2011, a microbial and enzymatic hybrid fuel cell used an anode consisting of a chitosan-MWCNT 3D macropores matrix electrode (Figure 59) [START_REF] Higgins | Hybrid Biofuel Cell: Microbial Fuel Cell with an Enzymatic Air-Breathing Cathode[END_REF]. The microbial anode was inoculated with culture of Shewanella MR1 and lactate as an electron donor. At the cathode, a carbonbased GDE with immobilized laccase was used for ORR by DET. The MFC achieved a power density of 26 W/m 3 and an open circuit potential of 1 V. This system was stable for at least 5 days, with a loss of only 4 % of the potential.

Figure 59 Schematic drawing of 3D Hybrid Fuel Cell. The flow-through anode compartment is filled with a highly porous chitosan-MWCNT composite anode material that was inoculated with Shewanella MR1. The enzymatic cathode is made from teflonized carbon black with adsorbed laccase as the bioelectrocatalysts. High-magnification SEM images show insight into both electrodes: (left) Shewanella MR1 in MWCNT and (right) the porous carbon black gas-diffusion electrode. [START_REF] Higgins | Hybrid Biofuel Cell: Microbial Fuel Cell with an Enzymatic Air-Breathing Cathode[END_REF] In another example, the Copper efflux oxidase from E. coli (MCO) was adsorbed on black-modified Ketjen carbon paper [START_REF] Kontani | Air diffusion biocathode with CueO as electrocatalyst adsorbed on carbon particle-modified electrodes[END_REF]. This biocathodic GDE was supplemented directly with O2 from the air and achieved ORR values of 20 mA•cm -2 at 0.2 V vs. SHE. Lalaoui et al., in 2015 developed an HFC based on GDEs and CNT [START_REF] Lalaoui | A membraneless air-breathing hydrogen biofuel cell based on direct wiring of thermostable enzymes on carbon nanotube electrodes †[END_REF]. CNT were covalently functionalized to enhance the immobilization of bilirubin oxidase (BOD) from Bacillus pumilus achieving high current densities up to 3 mA•cm -2 at 0.2 V vs. SHE for oxygen reduction from air. Then this cathode was used in a membrane-less hydrogen biofuel cell, together with an anode consisting of CNT functionalized for the immobilization of the hydrogenase from Aquifex aeolicus. The advantage of this biofuel cell was that the membrane-less design allowed for oxygen reduction at the cathode and oxidation of hydrogen at the anode without inhibition of hydrogenase by O2. Another example of HFC was reported in 2018 (Figure 60) [START_REF] Gentil | Oriented Immobilization of [NiFeSe] Hydrogenases on Covalently and Noncovalently Functionalized Carbon Nanotubes for H 2 /Air Enzymatic Fuel Cells[END_REF] where the [NiFeSe]-Hydrogenase from Desulfomicrobium baculatum was immobilized on covalent functionalized CNT electrodes, through hydrophobic interactions. By exploiting these interactions, the enzyme was able to work efficiently in an H2/air diffusion HFC, along with the BOD from Myrothecium verrucaria. Microbial Electrolysis Cells (MECs) work according to the same principle as MFCs.

Microbes oxidize organic substrates at the anode and molecules are electrochemically reduced at the cathode (such as CO2 and H + ) by microbes. Unlike MFCs, the flow of electrons, from the anode to the cathode, generally is supplied by an external power source (ΔG > 0) (Figure 58C). Cathodic reduction reactions are typically for Hydrogen Evolution Reaction (HER) and different hydrocarbons synthesis processes (such as CH4 and EtOH). MECs cathodes are generally microbial community (biofilm) which in addition to the desired reaction can carry out the synthesis of by-products. For example, in the case of HER it was shown that in the biofilm the hydrogenogens Desulfovibrio species were predominant [START_REF] Aulenta | Linking Bacterial Metabolism to Graphite Cathodes : Electrochemical Insights into the H2 -Producing Capability of Desulfovibrio sp[END_REF]. However, side reactions such as the reduction of CO2 to CH4 can be observed, in this the use of cathodic GDE is very useful, especially in single chamber MEC, facilitating the rapid removal and recovery of the produced H2, in order to minimize its microbial metabolism [START_REF] Horst | Application of Gas Diffusion Electrodes in Bioelectrochemical Syntheses and Energy Conversion[END_REF].

Tartakovsky et al., built and optimized a MEC for HER, based on the microbial oxidation of acetate, which operated continuously and electrically supplemented (1.16 V) [START_REF] Tartakovsky | Biocatalyzed hydrogen production in a continuous flow microbial fuel cell with a gas phase cathode[END_REF]) [START_REF] Tartakovsky | High rate membrane-less microbial electrolysis cell for continuous hydrogen production[END_REF]. The optimized MEC displayed a volumetric selective production of hydrogen was improved to 6.3 L per liter of anode chamber and day and the presence of methane in the outgoing gas was only 2.1 %.

The possibility of using MECs to perform CO2RR processes was first demonstrated in 2010, here the biofilm included acetogenic bacteria, such as Sporomusa spp. and

Clostridium spp. This graphite cathode supported biofilms were able to reduce CO2 to acetate and small amount of 2-oxobutyrate [START_REF] Nevin | Microbial Electrosynthesis : Feeding Microbes Electricity To Convert Carbon Dioxide and Water to Multicarbon Extracellular Organic Compounds[END_REF].

Electroenzymatic synthesis (EES) mostly refers to the possibility of functionalizing H2O2driven organic compounds such as selective epoxidation, hydroxylation and heteroatom oxygenation. These reactions are catalyzed by enzymes such as peroxygenases and peroxidases. The main problem for the industrial-scale applications of EES is the enzymatic instability towards the H2O2 co-substrate. Which should be generated electrochemically at a low but sufficient concentration (Figure 58D). Also, in EES the GDEs are fundamental for the availability of O2, an example of a cathode GDE-based for the conversion of phenolic monoterpenes (such as thymol and carvacrol), into antimicrobials has been described [START_REF] Getrey | Enzymatic halogenation of the phenolic monoterpenes thymol and carvacrol with chloroperoxidase[END_REF]. To date, it has never been developed an electrochemical cell for the H2 evolution from CO based on Hydrogenase and [NiFe]-CODH. Only one work shows the proof of concept where the enzymatic WGSR was tested in a narrow anaerobic condition in a glass tube filled with a suspension of ChCODH-I and Hydrogenase 2 from E. coli adsorbed on a graphite platelets. The reaction was started by injecting aliquots of CO into the gas phase of the glass tube, the CO and H2 concentrations were monitored over time by gas chromatography. The WGSR TOF was estimated to be at least 2.5 s -1 at 30 °C, which is based on the less active enzyme (Hydrogenase 2) and is much lower than the rate at which the hydrogenase can work [START_REF] Lazarus | Water -Gas Shift Reaction Catalyzed by Redox Enzymes on Conducting Graphite Platelets[END_REF].

EES processes have been exploited to carry out various reactions such as dehydrogenation, oxygenation, disproportion and formation of oxidative bonds [START_REF] Schmitz | Enzyme-Based Electrobiotechnological Synthesis[END_REF]. Furthermore, EES are still limited in terms of stability and final product yield [START_REF] Schmitz | Enzyme-Based Electrobiotechnological Synthesis[END_REF], this is mainly due to the fact that combining promising reaction engineering concepts with the required catalytic conditions of the enzyme is a high task. In the context of this project, attention will now be focused on the electrochemistry of [NiFe]-CODHs.

The first time that the methyl viologen mediated electroreduction of CO2 into CO by a

[NiFe]-CODH has been demonstrated was in 2003. The bifunctional enzyme (CODH/ACS) from Moorella Thermoacetica reduces CO2 at -0.57 V vs. SHE, with a Faradaic efficiency (see Annex) of almost 100 % at pH 6.3 [START_REF] Shin | Highly Selective Electrocatalytic Conversion of CO2 to CO at -0.57 V ( NHE ) by Carbon Monoxide Dehydrogenase from Moorella thermoacetica[END_REF]. After this work, the other studies focused on the monofunctional CODHs from C. hydrogenoformans, conducted by Armstrong and co-workers.

In 2013, they investigated the inhibition of the two ChCODHs (I and II) by protein film electrochemistry (PFE, see Annex). In general, enzymes were adsorbed nonspecifically on a PGE electrode or by using molecules that improve the stability of the protein film, such as polymyxin (V. C.-C. [START_REF] Wang | A unified electrocatalytic description of the action of inhibitors of nickel carbon monoxide dehydrogenase[END_REF] Wang, Ragsdale, et al., 2013).

(V. C.-C.
In addition to improving the surface coverage of the enzyme, the PGE electrodes were also functionalized with MWCNT by dip-coating a solution of 1 mg•mL -1 of MWCNT dispersed in DMF. The MWCNT were further functionalized with polymyxin (V. C.-C. [START_REF] Wang | Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase[END_REF]. DvCODH recovers all the activity while only 20 % at most was restored for ChCODH II [START_REF] Merrouch | O2 Inhibition of Ni-Containing CO Dehydrogenase Is Partly Reversible[END_REF]. The same research group also characterized the O2 inhibition of ChCODH IV. This enzyme shows a very high affinity for CO and is able to oxidize it at the diffusion limit over a wide range of temperatures (Km for CO at 25 °C of 8 ± 1 µM and 47 ± 3 nM for ChCODH II and ChCODH IV, respectively) [START_REF] Domnik | CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2[END_REF]. Indeed, its role in defense against oxidative stress has been proposed, which support the theory that CODHs can be different in terms of activity and physiological roles [START_REF] Domnik | CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2[END_REF]. The results shows that the high affinity of ChCODH IV for CO protects the enzyme from O2 inhibition. The two TcCODHs displayed behaviors similar to that of ChCODH IV [START_REF] Benvenuti | The two CO-dehydrogenases of Thermococcus sp. AM4[END_REF]. The results about these two different behaviors of O2 competition with CO reinforce the previous proposed theory on the different possible functions of CODHs. These works have highlighted the possibility of using CODH in bio-electrocatalytic CO2RR processes, in mild conditions, but also in bifunctional devices allowing the reduction of CO2 to CO coupled to H2 oxidation by a Hydrogenase. However, improvements are needed, such as the compartmentalization of the two enzymes on GDE electrodes, for example to prevent the CO inhibition of hydrogenases or improve direct electron transfer by co-immobilizing the two enzymes on the CNT. After the presentation of

[NiFe]-CODHs, the comparison with catalysts currently used in CO2RR processes such as Formate Dehydrogenase, metal-based catalysts and molecular catalysts will be presented in the next section.

To date, five oxidoreductases are described using naturally CO2 as substrate: Formate Dehydrogenase (FDH), Carbon dioxide reductase, remodeled nitrogenase, Pyruvate:Ferredoxin oxidoreductase (PFOR) and CODH. Some in vitro catalytic properties and products of these enzymes are shown in Table 14.

HCOO -+ NAD(P) + ↔ CO2 + NAD(P)H

FDHs are attractive enzymes for the production of formate through CO2 reduction.

However, CO2 reduction being on unfavorable endergonic reaction, most FDH catalyze the oxidation of formate [START_REF] Crable | Formate Formation and Formate Conversion in Biological Fuels Production[END_REF][START_REF] Førgaard | Classification and enzyme kinetics of formate dehydrogenases for biomanufacturing via CO2 utilization[END_REF]. But under appropriate thermodynamic conditions (notably a high substrate concentration) CO2 is reduced to formate. FDHs can be divided into metal independent (Type 1) and metal dependent (Type 2) catalysts. The latter can adopt different molecular organization, such as monomers, heterodimers or heterotrimers, while Type 1 are found as monomers. Type 1 FDHs usually catalyze the reduction of formate to CO2 using NAD + as a cofactor, while Type 2 possess active sites based on Molybdenum or Tungsten, Mo(VI)

or W(VI) as part of a pyranopterin guanosine dinucleotide cofactor and is coordinated to the proteins via cysteine or selenocysteine residues. Furthermore they also possess FeS clusters for the electron transfer [START_REF] Førgaard | Classification and enzyme kinetics of formate dehydrogenases for biomanufacturing via CO2 utilization[END_REF]. Metal-dependent enzyme are mostly O2 sensitive but are the most active FDH (Kcat > 1000 s -1 ) compared to the O2 insensitive low active metal independent ones (Kcat < 1 s -1 ) for formate oxidation. Soluble NADH & metal-independent FDHs can exhibit CO2 reduction activities (Kcat = 10 s -1 ), which is 10 times higher than metal-independent enzymes. Unlike CODHs, FDHs need more attention to electrode immobilization to achieve direct electron transfer, e.g., graphite-epoxy electrodes, CNT-coated graphite electrodes, TiO2 or polymyxin coated PGE electrodes [START_REF] Meneghello | Electrochemical studies of CO2reducing metalloenzymes[END_REF].

Electrocatalytically activity of FDHs has been studied in depth over the past decade by

Reisner's research group with the aim of using light as a sole source of energy and water as a sole source of electrons for driving the CO2 reduction to formate (photoreduction of CO2). In 2018 an electroenzymatic synthesis was developed system using a photoanode capable of catalyzing the oxidation of water to O2 and a cathode with FDH adsorbed on a hierarchically structured inverse opal titanium dioxide scaffold (Figure 65) [START_REF] Sokol | Photoreduction of CO 2 with a Formate Dehydrogenase Driven by Photosystem II Using a Semi-arti fi cial Z -Scheme Architecture[END_REF]. This system achieves the light-guided CO2 conversion to formate with a selectivity of 80 % and the FDH-functionalized cathode retain approximately 83 % of the initial activity after 2 hours.

Figure 65 Schematic representation of the semi-artificial photosynthetic tandem PEC cell coupling CO2 reduction to water oxidation. A blend of POs and PSII adsorbed on a dpp-sensitized photoanode (IO-TiO2|dpp|POs-PSII

) is wired to an IO-TiO2|FDH cathode (species size not drawn to scale) [START_REF] Sokol | Photoreduction of CO 2 with a Formate Dehydrogenase Driven by Photosystem II Using a Semi-arti fi cial Z -Scheme Architecture[END_REF] Based on the same principle, they used metal oxide electrodes (TiO2) for the immobilization of an FDH and RuP or diketopyrrolopyrrole (DPP) as photosensitizers (Figure 66). The latter injects photoexcited electrons into the conductive TiO2 which are transferred to the FDH which carries out the reduction of CO2 to formate with a TOF of 11 s -1 [START_REF] Miller | Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar-Driven Reduction of Carbon Dioxide[END_REF]. This system shows a stable formate production for about 6 hours and after 24 hours of continuous photocatalysis the residual enzymatic activity was about 36 % [START_REF] Miller | Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar-Driven Reduction of Carbon Dioxide[END_REF]. [START_REF] Miller | Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar-Driven Reduction of Carbon Dioxide[END_REF] The same group also used a conductive indium tin oxide (ITO) as an electron relay to connect the active sites of FDH and Hydrogenase (Figure 67B) [START_REF] Sokol | Reversible and Selective Interconversion of Hydrogen and Carbon Dioxide into Formate by a Semiarti fi cial Formate Hydrogenlyase Mimic[END_REF]. This system was bioinspired by the formate hydrogenlyase complex (FHL), which naturally catalyzes the oxidation of formate (FDH subunit) to the production of H2 (Hydrogenase subunit) (Figure 67A). This system shows reversibility and good stability for 24 hours with a selectivity for formate and H2 production of 76 and 77 %, respectively. Species size not drawn to scale [START_REF] Sokol | Reversible and Selective Interconversion of Hydrogen and Carbon Dioxide into Formate by a Semiarti fi cial Formate Hydrogenlyase Mimic[END_REF] In 2007, Armstrong and collaborators developed a model system for the photoreduction of CO2 to CO using visible light (Figure 68) [START_REF] Woolerton | Efficient and Clean Photoreduction of CO 2 to CO by Enzyme-Modified TiO 2 Nanoparticles Using Visible Light[END_REF]) [START_REF] Woolerton | CO2 photoreduction at enzyme-modified metal oxide nanoparticles †[END_REF]. They adsorbed ChCODH-I, as a catalyst, and a RuP, as a photosensitizer, on n-type MOx semiconductor nanoparticles (such as TiO2, ZnO, anatase, etc.). The working principle is as follows: visible light excites the RuP which injects electrons into the semiconductor, electrons are finally transferred through the FeS cluster of the CODH to the active site where the CO2 is reduced to CO. They also managed to regenerate the photosensitizer, through a sacrificial electron donor. This system in the best configuration achieved a TOF of 0.14 s -1 and TON of 2 000. This low TOF was improved using a RuP free system, where CODH was assembled on CdS nanocrystals with visible light harvesting capability, which offered sufficient driving force to reduce CO2 to CO.

This system achieved an average TOF of 1.23 s -1 and a TON of 2 200 [START_REF] Benniston | Visible light-driven CO2 reduction by enzyme coupled CdS nanocrystals[END_REF]. As previously mentioned, CO2RR is an important strategy to generate valuable chemicals from CO2. In recent years, CO2 to formate electrolysis cells have been reported based on FDHs. Mo-FDH was immobilized and electrically wired to a redox polymer consisting of cobaltocene grafted onto polyallylamine. The bioelectrode achieved the reduction of CO2 to formate with a Faradaic efficiency of 99 % at -0.66 V vs. SHE [START_REF] Yuan | Redox Polymers Creating a Low-Potential Redox Polymer for Efficient Electroenzymatic CO 2 Reduction[END_REF].

K. Kano and colleagues reported earlier the immobilization of W-FDH on Ketjen Black electrode functionalized with polytetrafluoroethylene groups in order to promote the immobilization of W-FDH through hydrophobic interactions. The bioelectrode reached a current density of about 20 mA•cm -2 under atmospheric pressure and ambient temperature in neutral pH [START_REF] Sakai | Electrochemistry Communications Ef fi cient bioelectrocatalytic CO 2 reduction on gas-diffusiontype biocathode with tungsten-containing formate dehydrogenase[END_REF].

CO2-to-CO electrolyzers with high efficiency and high specificity is an important strategy for CO2 valorization. This type of electrolyzers must operate at high current density of several hundreds of mA cm -2 . Nowadays, this types of electrolyzers are based on gold and silver catalysts operating in gas-diffusion electrolyzers [START_REF] Torbensen | Emergence of CO2 electrolyzers including supported molecular catalysts[END_REF] In another example, a cobalt porphyrin catalyst was used as a building block to synthetize a covalently-linked macrostructure (Covalent organic framework or COF) [START_REF] Lin | Covalent organic frameworks comprising cobalt porphyrins for catalytic CO 2 reduction in water[END_REF]. Another Co-based complex has recently been reported, derived from phenanthroline subunits an N4-macrocyclic Co complex (Co-CPY) has been synthetized

(Figure 69C) (Sun et al., 2020). Other molecular catalysts based on earth-abundant metals have also been synthesized and have demonstrated selective CO2RR such as a pyrene-substituted iron porphyrin (FePhpyr) (Figure 69D) (Maurin & Robert, 2016) and a pyrene-substituted manganese-bipyridine-CO complex [MnBr(2,2'-bipyridine)(CO)3]

(Figure 69E) (Reuillard et al., 2017).

Among these catalysts which have been implemented in pre-industrial flow cells operating in alkaline media at CO2-diffusing electrodes, several CNT-supported cobalt phthalocyanine have reached current densities >150 mA•cm -2 with near to 100% CO Faradaic efficiency [START_REF] Ren | Molecular electrocatalysts can mediate fast, selective CO 2 reduction in a flow cell[END_REF](M. [START_REF] Wang | CO2 electrochemical catalytic reduction with a highly active cobalt phthalocyanine[END_REF]. Furthermore, stability and Faradaic efficiency were kept for at least 8-10 hours at lower current densities (20-75 mA•cm -2 ). In addition, other metal-based catalysts have been synthesized such as:

Copper polyphthalocyanine (CupolyPc) (Karapinar et al., 2020), gold nanoneedles or nanowires (M. Liu et al., 2016) [START_REF] Zhu | Active and Selective Conversion of CO 2 to CO on Ultrathin Au Nanowires[END_REF], palladium nanoparticles (D. [START_REF] Gao | Size-Dependent Electrocatalytic Reduction of CO 2 over Pd Nanoparticles[END_REF], and nanoporous silver [START_REF] Lu | A selective and efficient electrocatalyst for carbon dioxide reduction[END_REF]. The performances of several best metalbased catalysts are summarized in Table 15. [START_REF] Wang | CO2 electrochemical catalytic reduction with a highly active cobalt phthalocyanine[END_REF]; 2 [START_REF] Ren | Molecular electrocatalysts can mediate fast, selective CO 2 reduction in a flow cell[END_REF]; 3 [START_REF] Kramer | Polymer coordination promotes selective CO2 reduction by cobalt phthalocyanine[END_REF]; 4 [START_REF] Takanabe | Simultaneous Reduction of CO 2 and Splitting of H 2 O by a Single Immobilized Cobalt Phthalocyanine Electrocatalyst[END_REF]; 5 (X. [START_REF] Zhang | Highly selective and active CO 2 reduction electro-catalysts based on cobalt phthalocyanine/carbon nanotube hybrid structures[END_REF]; 6 (M. Wang et al., 2018); 7 [START_REF] Lin | Covalent organic frameworks comprising cobalt porphyrins for catalytic CO 2 reduction in water[END_REF]; 8 (Karapinar et al., 2020); 9 (M. Liu et al., 2016); 10 [START_REF] Zhu | Active and Selective Conversion of CO 2 to CO on Ultrathin Au Nanowires[END_REF]; 11 (D. [START_REF] Gao | Size-Dependent Electrocatalytic Reduction of CO 2 over Pd Nanoparticles[END_REF]; 12 [START_REF] Lu | A selective and efficient electrocatalyst for carbon dioxide reduction[END_REF]; 13 (Maurin & Robert, 2016); 14 (Reuillard et al., 2017) The understanding of complex Ni enzymes and the development of bio-inspired systems for CO2 reduction were the two major driving forces that led us to start the study of the [NiFe]-CODH from Rhodospirillum rubrum (RrCODH) and allowed us to obtain the following results. The choice of RrCODH is based on the following criteria: the enzyme has been thoroughly characterized by a combination of approaches, its X-ray structure has been solved in 2001, the Ni chaperones involved in its maturation are well known and characterized, and finally, its specific activity for CO oxidation in solution is the highest under ambient conditions compared to that of the other described CODHs.

-bipy = [MnBr(2,2'-bipyridine)(CO)3] ; 1 (M.
When I started my PhD three years ago, several aims were raised. The first was to provide a novel simple and efficient protocol for the heterologous production of an active recombinant RrCODH with a high production and purification yields, in order to facilitate both its detailed characterization and the development of it use in CO2RR processes.

The second objective was to provide insights on the maturation processes of [NiFe]-CODHs. For this reason, before this works our group together with international collaborations, has already characterized the two Ni chaperones CooT and CooJ. These latter proteins are widely spread in the genome of several microorganism that also possess the CODH and CooC genes [START_REF] Darrouzet | Revisiting the CooJ family , a potential chaperone for nickel delivery to [ NiFe ]carbon monoxide dehydrogenase[END_REF] (Alfano et al., 2018) [START_REF] Timm | The CO dehydrogenase accessory protein CooT is a novel nickel-binding protein[END_REF]. The ultimate goal was to show whether RrCODH could be electrochemically connected to an electrode, since almost no other electrochemical studies on RrCODH had been performed prior to this study. Furthermore, with the aim of a future development of functional CO2RR devices based on RrCODH, we have investigated the best electrode surface immobilization technique to obtain electrochemical performances at least comparable to non-biological CO2RR catalysts. In support of this last point, our laboratories have already succeeded in the efficient electrochemical immobilization of other redox enzymes (Hydrogenase, Peroxidase, Glucose Oxidase, exc.) on carbon nanotubes, exploiting the advantages of using these nanomaterials.

■ Providing a novel method for the heterologous production of a fully active recombinant RrCODH with high yields.

The protocol provided for the aerobic production of Rec-RrCODH in E. coli has been extensively studied and validated prior to publication. I spent about a year of this PhD project discovering the best conditions and problems related to aerobic production and anaerobic purification, with the aim of keeping a stable and fully active enzyme without protein or metallic co-factors damages, such as proteolysis, oxidative reactions, due to the interaction of reducing agents (DTT and DTH) with O2.

Regarding the production yield, the following table summarizes the reported growth conditions of the host bacteria together with the yields of crude microbial culture and pure CODH enzymatic preparations.

CODH

Host bacteria Growth conditions Cells Yield (g L -1 ) Purification yield (mg L -1 ) Reference

Ch II

E. coli

Fully anaerobic expression, N2 gas supply. Dv CooC ND 0.3-0.6

Rr from native R. rubrum

Fully anaerobic expression in 80L Fermenter light and gas (N2 and CO) supply.

7.5 0.3-3 [START_REF] Bonam | Purification and characterization of carbon monoxide dehydrogenase, a nickel, zinc, iron-sulfur protein, from Rhodospirillum rubrum[END_REF] RecRr

E. coli

Fully aerobic auto induced expression in standard Erlenmeyer, 3 and 21 hours respectively at 37 and 15 °C.

12-20 14-17

This work

RecRr CooCTJ 4-6 107

Our expression system requires no specific precautions for E. coli growth, such as fermenter or gas supply. The E. coli cells yield is approximately 4-7 times higher than that reported for ChCODH II, while the yield of pure enzyme (RecRrCODH) is approximately 28-170 and 7-10 times higher than DvCODH and ChCODH II preparations, respectively.

These high yields of RecRrCODH preparations allowed for several fine biochemical, spectroscopic and electrochemical characterizations presented above, in particular the EPR titrations, that is notoriously expensive in baculatum [START_REF] Gentil | Oriented Immobilization of [NiFeSe] Hydrogenases on Covalently and Noncovalently Functionalized Carbon Nanotubes for H 2 /Air Enzymatic Fuel Cells[END_REF]. S1 Supporting Information).

Cloning and expression

The Rhodospirillum rubrum cooS gene was obtained from DNA 2.0 in pET15b as a codon-optimized gene for expression in E. coli (640 residues, 66911 Da). The cooS gene was subcloned in a modified version of the vector pCDFDuet-1, namely pCDF-Duet1-LIC1 (Structural Genomics Consortium, Toronto) which contains a TEV cleavage site downstream the histidine tag at the N-terminus (662 residues, 69547 Da). The Rhodospirillum rubrum cooCTJ genes, manually codon optimized genes for expression in E. coli were obtained from Genscript in pBAD24 vector (NcoI/HindIII):

pBAD_cooCTJ. The cooCTJ genes were subcloned into pRSFDuet1 (NcoI/HindIII) to

give the pRSF_RrcooCTJ construction.

E. coli BL21 (DE3) cells transformed with pCDF-Duet1-LIC1_RrcooS alone or cotransformed with pRSF_RrCooCTJ were cultured under aerobic conditions in autoinduction medium, supplemented with appropriate antibiotics, 1 mM Fe(II)SO4 (Sigma Aldrich), 2 mM L-Cysteine (Sigma Aldrich) and 500 µM NiSO4 (ACROS ORGANICS)

for 3 hours at 37 °C and 180 rpm (OD600 0.8-1.5) and subsequently transferred for 21 hours at 15 °C and 180 rpm (OD600 9-14). Cultures were centrifuged and cell pellets collected under argon flow before being stored in liquid nitrogen.

calculated for each sample. Protein concentration in all samples was determined by integration of the differential refractive index (dRI) peak after injection of 20 µL protein.

Metal content determination

Bathophenantroline assay was used for the determination of Fe(II) by following a previous published protocol. 1 Ammonium iron(II) sulfate (Sigma Aldrich) was used for made the calibration curve between 0 to 215 µM Fe(II). RecRrCODH and RecRrCODH CTJ were diluted between 2.8 to 0.36 µM of dimer and each dilution was measured in triplicate. The absorbance 535-680 nm was measured by using Varian Cary® 50 UV-Vis Spectrophotometer (Agilent Technologies).

4-(2-pyridylazo)resorcinol (PAR) assay was used for the determination of Ni(II) by following a previous published protocol. 2 Nickel(II) sulfate heptahydrate (Sigma Aldrich)

was used for the calibration curve between 0 to 40 µM Ni(II). RecRrCODH and RecRrCODH CTJ in buffer C (see above) were preincubated 15 minutes with 1 mM EDTA to remove aspecific Ni(II) traces, after the buffer was exchanged with 50 mM TrisHCl buffer pH 8.5 by using Micro BioSpin® 6 columns (Bio-Rad). The CO oxidation activity was tested before and after the EDTA treatment to verify the integrity of the enzyme.

RecRrCODH and RecRrCODH CTJ were diluted between 9.6 to 1.1 µM of dimer and each dilution was measured in triplicate. The absorbance was measured at 495 nm by using Varian Cary® 50 UV-Vis Spectrophotometer (Agilent Technologies).

CO oxidation activity measurements

Rec-RrCODH and Rec-RrCODH CTJ were diluted to about 20 and to about 5 nM of monomer, respectively. CO oxidation activity was assayed at 25 °C by following the reduction of methyl viologen at 604 nm [ε=13.6 mM -1 cm -1 ] in CO-saturated solutions containing 12 mM methyl viologen and 1 mM DTT (standard conditions). CODH specific activity unit is expressed as micromoles of CO oxidized per minute per milligram of protein. The standard buffer used to dilute the enzyme prior to measurements was 50 mM TrisHCl pH 8.5, 5 mM DTT, 1 mM DTH (buffer A). For pH dependence measurements (Figure 2 A), Rec-RrCODH CTJ was diluted in buffer A at 21 °C. CO oxidation activity was measured in standard conditions, except that the following range of pH and buffers were used: 100 mM NaAcetate for pH 4.0 or 5.0 and 50 mM BisTris Propane from pH 6.0 to pH 11.5. For temperature dependence measurements (Figure 2 B), Rec-RrCODH CTJ was diluted in solution A at 21 °C and the activity was measured in standard conditions except that the following range of temperatures was used: 21, 25, 30, 35, 40, 45, 50, 55 and 60 °C. For stability experiments (Fig. 2 CD), Rec-RrCODH CTJ was diluted in buffer A, except that the range of pH and buffers described above was used at 21 °C.

The activity was measured at t0 and t24h. Rec-RrCODH CTJ was diluted in solution A and incubated at different temperatures (from 21 to 60°). The activity was measured at t10, t30 and t60 min.

Redox titration by Electron Paramagnetic Resonance

Purified Rec-RrCODH CTJ at 108 µM was oxidized by buffer exchange with 0.1 M MOPS pH 8.0 using a desalting a NAP™-10 column (GE Healthcare) and diluted to 70 µM (dimer concentration).

Enzyme preparations were titrated in glove box in the presence of the following mediators

Electrochemical measurements

1-pyrenebutyric acid adamantyl amide was prepared as previously described. 3 All reagents were purchased from Sigma Aldrich. Commercial grade thin Multi-Walled Carbon Nanotubes (MWCNT, 9.5 nm diameter, purity > 99%). Carbon nanomaterials were used as received without any purification. When not used, the enzymes were stored at 4 °C. All the reagents were used without further purification. All solvents were of analytical grade. Distilled water was passed through a Milli-Q water purification system.

The electrochemical experiments were carried out in a three-electrode electrochemical gas thigh cell using a Biologic VMP3 Multi Potentiostat inside an anaerobic glove box (O2 <2 ppm, Jacomex). The MWCNT bioelectrodes were used as working electrodes. Pt Robinson buffer pH 5.0. For information, CO solubility in water is 3.3 mL and 2.3 mL per 100 mL at 0°C and 20°C, respectively. CO2 solubility in water is 171 mL and 88 mL per 100 mL at 0°C and 20°C, respectively.

Preparation of the glassy carbon-and gas diffusion-modified MWCNT electrode and enzyme immobilization

The working electrodes were glassy carbon and gas diffusion electrodes (3 mm diameter).

N-methyl-2-pyrrolidinone (NMP) dispersion of MWCNTs were prepared by 30 minutes sonication of 5 mg MWCNTs dispersed in 1 mL NMP until homogeneous black suspension was obtained. Then 20 µL of the MWCNTs solution were drop-casted on a GCE/GDE and NMP was removed under vacuum leaving a 5-µm-thick film on the GCE.

The functionalised electrodes were then incubated for 4 hours with 25 µL of the enzymatic Rec-RrCODH and Rec-RrCODH CTJ solutions (19 µM dimer concentration pH 8.5) which were pre-reduced in presence of 5 mM DTH and 5 mM DTT. The latter step was carried out inside an anaerobic glove box (O2 <2 ppm, Jacomex). The electrodes were finally washed with buffer TrisHCl 50 mM pH 8.5. When not used the electrodes were kept in buffer TrisHCl 50 mM pH 8.5.

Functionalisation of MWCNT with 1-pyrenebutyric acid adamantyl amide and enzyme immobilization

MWCNT-modified electrodes were soaked for 30 minutes in DMF solution containing 10 mM 1-pyrenebutyric acid adamantyl amide and after rinsed in DMF solution and two times in water. The functionalised electrodes were then incubated for 4 hours with 25 µL of the enzymatic Rec-RrCODH and Rec-RrCODH CTJ solutions (19 µM dimer concentration pH 8.5) which were pre-reduced in presence of 5 mM DTH and 5 mM DTT. The latter step was carried out inside an anaerobic glove box (O2 <2 ppm, Jacomex).

The electrodes were finally washed with buffer TrisHCl 50 mM pH 8.5.

Gas Chromatography products analysis of MWCNT ADA immobilized RecRrCODH CTJ

RecRrCODH CTJ immobilized on either MWCNT ADA glassy carbon-or gas diffusionelectrodes were used to analyze the electrocatalysis products. The production of CO and CO2, from CO2 and CO, respectively were analyzed with a PerkinElmer Clarus 500 gas chromatography (GC). Chronoamperometry experiments were performed under a saturated and continuous flow of each substrate. Regarding the glassy carbon electrode, an aliquot of the gas phase from the gas-tight electrochemical cell was taken and injected into the GC. While for the gas diffusion electrode the outgoing gas was taken and directly injected into the GC. Controls experiments were performed with injections of CO (10 µL of 100 % CO) and CO2 (50 µL of a mixture 20 % CO2 80% N2) gasses.

The results presented in Chapter 2 strongly encouraged us to continue to improve the electrochemical performances of recombinant RrCODH. As presented in Chapter 1, nonspecific enzymatic immobilization on the electrode surface is useful for starting to study a redox enzyme and avoid drawbacks such as partial unfolding of the biocatalyst.

However, the "soft" immobilization of the enzyme often results in poor stability over time, as shown in Chapter 2 for the RecRrCODH CTJ by chronoamperometry under CO2.

Another bottleneck of non-specific immobilization is the inability to control the orientation of the catalysts on the electrode surface.

For these reasons we decided to study the possibility of immobilizing the recombinant RrCODH via affinity interactions in order to improve its stability over time and to better control the orientation of the enzyme at the electrode surface. Our strategy was based on the affinity immobilization provided by the nitrilotriacetic acid (NTA) chemistry, as previously shown for glucose oxidase, laccase and nitrate reductase [START_REF] Campbell | Affinity binding via zinc(II) for controlled orientation and electrochemistry of histidine-tagged nitrate reductase in self-assembled monolayers[END_REF]) [START_REF] Sosna | A His-tagged Melanocarpus albomyces laccase and its electrochemistry upon immobilisation on NTA-modified electrodes and in conducting polymer films[END_REF] (Holzinger et al., 2011) [START_REF] Balland | Oriented immobilization of a fully active monolayer of histidine-tagged recombinant laccase on modified gold electrodes[END_REF] [START_REF] Haddour | Electrogeneration of a poly(pyrrole)-NTA chelator film for a reversible oriented immobilization of histidine-tagged proteins[END_REF]. This electrode functionalization strategy allows the formation of oriented and fully electroactive enzymatic monolayer. Initially, NTA was developed for the purification of engineered proteins via the formation of specific metal coordination interactions between NTA and histidine-tagged recombinant proteins.

We

Materials and

Instruments. 1-pyrenebutyric acid adamantyl amide was prepared as previously described. [1] All reagents were purchased from Sigma Aldrich. Commercial grade thin Multi-Walled Carbon Nanotubes (MWCNT, 9.5 nm diameter, purity > 99%).

Carbon nanomaterials were used as received without any purification. RecRrCODH was co-produced in presence of the three Ni-chaperones (RrCooC, RrCooT and RrCooJ) and isolated as previously described. [2] When not used, the enzymes were stored at 4 °C. All the reagents were used without further purification. All solvents were of analytical grade. Distilled water was passed through a Milli-Q water purification system. Jacomex). The surface of GC electrodes was polished with a 2 μm diamond paste purchased from Presi (France) and rinsed successively with water, acetone, and ethanol.

A Pt wire placed was used as counter electrode, and the SCE or Ag/AgCl served as reference electrodes.

Synthesis of the ligands

Synthetic procedures for compound 1 and 2:

Addition of 1-Pyrenylmethyl bromide (555mg, 1.87mmol) to a solution of tacn orthoamide (260 mg, 1.87 mmol) in tetrahydrofuran (6 ml) produced a precipitate almost immediately. Stirring was continued for another 30 minutes after which the product was filtered and washed with absolute ethanol (2 x 2 ml) and ether (3 x 2 ml).

The green solid was then dissolved in water (6 mL) and heated at reflux for 4 hours. The solution pH was adjusted to 12 with NaOH, the product was extracted into chloroform (4 x 10 ml), the extracts dried over magnesium sulfate and the solvent removed under reduced pressure to give 1 (411 mg, 87 % yield).

Electrochemical analysis

The Langmuir-Freundlich model was employed to fit the experimental data, according to equation 1: [3,4] %𝐿𝑜𝑠𝑠

𝑒𝑞 = %𝑙𝑜𝑠𝑠 𝑚𝑎𝑥 × (𝐾 𝐼𝑚𝑖𝑑 𝑎𝑝𝑝 × [imidazole]) 𝑛 1 + (𝐾 𝐼𝑚𝑖𝑑 𝑎𝑝𝑝 × [imidazole]) 𝑛
Where %Losseq is the percentage of CO activity loss at the equilibrium, %lossmax is the maximum percentage of CO activity loss at maximum imidazole concentration, KImid app is the apparent association constant in water between imidazole and AcPyTACN sites at the modified electrode and n is Langmuir-Freundlich coefficient number. Table 1 shows the Langmuir-Freundlich model parameters obtained from fitting curves from Figure 4A. The achieved current densities for CO2 reduction showed no major differences (Table A1), confirming that the TACN moiety can chelate other metal ions and maintain its specificity for His-RecRrCODH immobilization. The modified electrodes exhibit electrocatalytic activity even if in very diluted (about 0.2 mg•mL -1 ). However, current densities are not high enough to conclude on a positive effect of the TACN-Ni anchoring sites towards specific immobilization of the CODH from the cell lysate. There might be competition between the histidine tagged RecRrCODH and other proteins which naturally possess a histidine tag, such as the co-expressed CooJ. The electrodes showed an average current density for CO2 reduction of 0.44 ± 0.11 mA•cm -2 (triplicate). These results confirm the strong affinity of the TACN-Ni moiety towards histidine-tagged proteins. To further confirm the strong affinity of AcPyTACN-Ni with histidine-tagged proteins, we also tested the immobilization of commercial Horseradish Peroxidase (HRP). HRP was obtained from Sigma Aldrich. The enzyme is a 44 kDa monomer that contains 18% glycosylated amino acids. HRP was posttranslational modified in order to chemically attach histidine residues to the carbohydrates surrounding the protein, as previously described [START_REF] Blankespoor | Dense Monolayers of Metal-Chelating Ligands Covalently Attached to Carbon Electrodes Electrochemically and Their Useful Application in Affinity Binding of Histidine-Tagged Proteins[END_REF]. The chemical modification consists in a first oxidation of the carbohydrates in As expected, the current density for H2O2 reduction is higher in the case of NTA-Ni However, the [2Fe2S] D-and the [4Fe4S] B-clusters are not affected by oxidative damage [START_REF] Wittenborn | Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase[END_REF]. To support the hypothesis of an increase in O2 tolerance in DvCODH, a C45G/T50C mutant, corresponding to the replacement of the C-X2-C motif by a C-X7-C motif, was constructed. The crystal structure of the C45G/T50C mutant, solved at 1.84 Å, revealed a canonical [4Fe4S] D-cluster (Figure 2B) although its overall structure is nearly identical to that of the WT enzyme (with an rmsd of 0.27 Å for 1250 their Cα atoms) and the region surrounding the D-cluster is not affected by the amino acid substitution (Figure 2A). The impact of O2 on WT and C45G/T50C enzymes on the catalysis as well as the protein structure and metal center were studied. Anaerobically grown crystals of the anaerobically purified C45G/T50C mutant were exposed to air as a function of time. The crystal structure collected immediately after exposure to air showed an intact

[4Fe4S] D-cluster (Figure 3A), while after 2 hours, the D-cluster begins to decay as shown by the electron density which probably corresponds to a mixture of conformational states and result in the poor fit of the [4Fe4S] cluster with 30% occupancy (Figure 3B) [START_REF] Wittenborn | The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni-Fe Carbon Monoxide Dehydrogenase[END_REF]. After 2 days under air, the crystal structure does not longer show any electron density related to the presence of a FeS cluster (Figure 3C) [START_REF] Wittenborn | The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni-Fe Carbon Monoxide Dehydrogenase[END_REF]. These results show that the C45G/T50C mutant is less stable towards air than the WT with a complete decomposition of the D-cluster in 2 days, although the B-and C-clusters are still intact (Figure 3DE). [START_REF] Wittenborn | The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni-Fe Carbon Monoxide Dehydrogenase[END_REF].

-Results

First, we designed the G44C/C49T RrCODH mutant (G44C/C49T) as a mimic of the WT DvCODH to change the C-X7-C binding motif to a C-X2-C one (Figure 4). We also designed single and double mutants of the two cysteines that coordinate the D-cluster: C41S, C49S, C49T, C41A/C49S and C41S/C49S (Figure 4).

-RrCODH D-cluster mutants are produced in E. coli under Aerobic and Anaerobic conditions.

The designed RrCODH mutants and the RrCooC, RrCooT, and RrCooJ accessory proteins were co-produced aerobically in E. coli following our previous established protocol (Chapter 2). Our standard anaerobic protocol was used for the purification of all mutants and led to pure enzymes in one step, with the exception of G44C/C49T which was difficult to concentrate, suggesting a loss of the homodimeric organization. For this mutant, several protocols were then tested in order to optimize its purification. G44C/C49T expression was then tested in the Rosetta2 ΔiscR strain (aG44C/C49T ΔiscR ), where the isc operon is constitutively expressed to ensure the optimum biogenesis of FeS clusters.

Furthermore G44C/C49T was produced under anaerobic conditions namely anG44C/C49T, following a method previously described for the recombinant ChCODHs (J. H. Jeoung & Dobbek, 2007) [START_REF] Domnik | CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2[END_REF]. However, despite these optimizations, G44C/C49T still remains difficult to produce and purify, in comparison to the other mutants.

-SEC-MALLS-RI.

SEC-MALLS-RI experiments were performed to determine the homogeneity and oligomerization state of the produced RrCODH mutants (Figure 5). The percentage of homodimer and monomer were determined by comparison of each concentration versus the total concentration of injected sample. The results are reported in Table 1. These results revealed an unexpected behavior of the different mutants: First in the case of Cys49 mutants, the mutation of Cys into Thr (corresponding to the replacement of the thiol group by a methyl group) seems to have a greater impact on the enzyme stability than the mutation of Cys to Ser (corresponding to the replacement of the thiol group by a OH group). Second, the removal of all coordinating-cysteines in C41S/C49S does not impact the dimerization, while the single C49S mutation does. Third, the G44C/C49T stability seems to be greatly increased by its expression in Rosetta2 ΔiscR strain.

To go further, we investigated the possible role of oxygen on the oligomerization state of the enzyme Indeed, in SEC-MALLS-RI experiments, each sample was exposed to air for about 30 minutes (corresponding to the duration of the experience), which could have a great impact on the conformation of the enzyme. To answer this hypothesis, SEC-MALLS-RI experiments were used to study the behaviour of both WT and C49T enzymes exposed to air for different incubation times (Figure 6). From the chromatograms in Figure 6, different oligomerization peaks can be observed:

279-220 ± 2 kDa tetramer/trimer, 139.6 ± 2 kDa dimer, 71.8 ± 4 kDa monomer. The oligomerization percentage results from Figure 6 are shown in Table 2. In both WT and C49T, the homodimeric state does not appear to be greatly impacted by air exposure and does not lead to the dissociation into monomers. However, some precipitation starts to appear at 1 h and increase with time. Therefore, the presence of mixture of monomers and dimers in the different mutants does not seem to be related to their O2 sensitivity. However, these experiments have to be conducted on the other mutants.

-UV-visible spectroscopy.

All mutants displayed the typical brown color due to the presence of [4Fe4S] clusters, with the exception of the three G44C/C49T samples which were colorless compared to the others enzymes at the same concentration. The presence of [4Fe4S] clusters was also investigated by UV-visible spectroscopy performed on oxidized enzymes (Figure 7).

The typical shoulder of the [4Fe4S] clusters and the purity coefficient (A420/A280) are

shown in Table 3. The purity coefficient relative to the native enzyme can be used to estimate the percentage of [4Fe4S] clusters together with the determination of the iron content (Table 3). A single mutation appears then to be sufficient to lead to the loss of the coordination of the D-cluster, based on the lower than expected Fe(II) content of all mutants. In addition, the lower purity coefficient and Fe(II) content of C49S, C41S, C41S/C49S also suggest that even B-or C-clusters could be lost or converted to [2Fe2S] clusters. In the case of G44C/C49T and C41A/C49S, the UV-visible spectra revealed the total The stability of the different mutants towards O2 was investigated by UV-visible spectroscopy. Samples of oxidized WT, C49T, C41S and C41S/C49T were anaerobically prepared and then exposed to air over time and UV-visible spectra were recorded every 15 minutes, in order to characterize the degradation kinetics of FeS clusters (Figure 8A). Then, semiquantitative analyses of the UV-visible spectra were performed using the software Fit-FeS v 1.1.1 (Figure 8B) [START_REF] Betinol | Spectral decomposition of iron-sulfur clusters[END_REF]. As predicted by the Fe(II) content, the mutants showed an initial [4Fe4S] content lower than the WT enzyme, but in all cases the degradation kinetics after 90 minutes under air are comparable between WT, C41S and C41S/C49S, although WT is stable during the first 15 minutes. Surprisingly C49T appeared to behave differently starting from a lower initial [4Fe4S] content and reaching the same content of WT at the end of the experiment. Similar results were also obtained for the [2Fe2S] clusters formation, a well described intermediate in the degradation process of [4Fe4S] clusters. From these first results, we can conclude that the removal of S-donors ligands induces as expected the loss of the coordination of Dcluster leading to an instability of the other FeS clusters and affecting the dimerization state of some mutants.

-In Solution and Electrochemical CO oxidation and CO2 reduction activities.

To determine whether these mutants are still able to catalyze the CO-to-CO2 interconversion, their catalytic efficiencies were investigated in solution and electrochemically. First, the activity in solution was measured for all enzymatic preparations. The enzymes were diluted in 50 mM TrisHCl, pH 8.5, 5 mM DTT and 1 mM DTH at 21 °C, then the CO oxidation activity was assayed in CO saturated 50 mM TrisHCl, pH 8.5, 1 mM DTT 12 mM methyl viologen at 25 °C (Table 4). electrodes. An investigation of the Cred1/Cred2 reversible redox system was first performed under argon (Figure 9). It is interesting to note that for all mutants it was possible to observe the non-turnover redox signal and also to estimate the surface coverage (Γmax) (Table 5). Due to the low Ni content, it is difficult to interpretate CV in non-turnover conditions, C49T exhibits similar midpotentials as compared to WT. Despite the high Ni content of C41S/C49S, the non-turnover signal is negligible, indicating that electron relays and/or C clusters are non-conserved or damaged in the protein. To further confirm this, a MWCNT AcPyTACN electrode was functionalized with C49T

following our previous described protocol (Chapter 3). The CVs were performed in CO and CO2 saturated solutions under continuous gas flow and agitation (Figure 11). C49T supports well the histidine tag-induced covalent immobilization on the MWCNT AcPyTACN electrode, indicating that the loss of D-cluster did not affect the N-terminus of the recombinant enzyme, as further confirmed by SEC-MALLS-RI oligomerization. Furthermore, current densities and TOF shows a similar trend to that of the C49T immobilized on the MWCNT ADA electrode (Table 6).

-Crystallization of RecRrCODH.

In order to further characterize the D-cluster mutants of the RrCODH we begin a crystallographic study of the WT RecRrCODH. The only structure of RrCODH available to date (at 2.8 Å resolution) has been obtained by microcapillary batch diffusion method under argon with precipitating agents: 10 % polyethylene glycol 8000 (PEG 8k), 0.1 M TrisHCl, pH 7.5, 0.4 M calcium chloride (CaCl2), 5 % 2-methyl-2,4-pentanediol (MPD). The RrCODH was about 20 mg•mL -1 in a fist approach, we screened similar crystallization conditions by using the hanging drop vapor diffusion method under argon in a glove box (Jacomex, O2 < 2 ppm) for RecRrCODH with or without His tag. However, the histidinetagged enzyme did not yield any nucleation. Thus, only the results obtained with the enzyme where the His tag was removed will be presented. Table 7 shows the results of the first crystallization test. These first results show that for almost all tested conditions, fast protein precipitation occurred. Then in order to slow down the protein precipitation, we decided to decrease the concentration of PEG 8k and CaCl2 as shown in Table 8. Therefore, to confirm the possibility of crystallizing the RecRrCODH, new assays were performed with a freshly purified enzyme. The conditions were screened according to the ones described in Table 10 and the best results are reported in Table 11. The results obtained with the A4, B4 and C4 conditions of Table 11 confirm Hence, if we take into considerations the results of the C49T mutant which showed a Fe(II) content and UV-visible absorption spectra consistent with the loss of D-cluster, the homodimeric organization appears to be unaffected which excludes a role of D-cluster in the stabilization of [NiFe]-CODHs dimers. This is also supported by high interface areas of 3846, 3469 and 3756 Å 2 observed in the crystal structures of RrCODH, ChCODH and DvCODH, respectively. Furthermore, C49T also displays a CO oxidation activity in solution and direct electrochemical CO/CO2 interconversion when immobilized on the functionalized MWCNT surface. All these results show that it is possible to observe an electron transfer directly from the B-clusters to the mediator or MWCNT. This is in line with the minimal distance of 10 Å between the B-cluster and the surface of the protein allowing electron tunnelling. If we then consider a linear correlation between Ni content and catalytic activities (Figure 13A), the performance of C49T is lower than expected from its Ni content, in solution: 4 271 U•mg -1 vs. a theoretical value of ~7 881 U•mg -1 considering 1.2 Ni/dimer and for the immobilized enzyme: 1.3 mA•cm -2 vs. a theoretical value of 2.2 mA•cm -2 . However, some mutants, such as C49T and C41A/C49S, appear to be less affected by the removal of the D-cluster, based on the normalized ratio of Ni content to CO2RR activity, which showed approximately 60 % of expected specific activity (Figure 13B). This hypothesis, in addition to the lower TOF, suggest that the D-cluster is dispensable, although its presence improves the catalytic efficiency of the enzyme. These results are reminiscent of the work performed with multicopper oxidases. In several examples, it has been shown that the type 1 copper site in MCOs, responsible for the electron relay to the trinuclear copper centre, could be bypassed or suppressed. Without this relay, the MCOs were still able to perform the reduction of O2 into H2O by directly connecting the trinuclear copper centre at the electrode [START_REF] Gentil | Efficiency of Site-Specific Clicked Laccase-Carbon Nanotubes Biocathodes towards O2 Reduction[END_REF] [START_REF] Sekretareva | Electron Transfer to the Trinuclear Copper Cluster in Electrocatalysis by the Multicopper Oxidases[END_REF] [START_REF] Scheiblbrandner | Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells[END_REF] (Lalaoui, Holzinger, et al., 2016) [START_REF] Dagys | Oxygen electroreduction catalysed by laccase wired to gold nanoparticles via the trinuclear copper cluster[END_REF]. Regarding the role of the D-cluster in O2 protection, it is difficult to conclude on this part since the G44C/C49T mutations did not yield a mutant with a [2Fe2S] D-cluster, supposed to increase the stability of the enzyme. However, our preliminary results show that O2 has an impact on the overall stability of the enzyme, as observed in SEC-MALLS-RI experiments. Conversely, the [4Fe4S] cluster content in C49T is visibly not strongly impacted by O2, compared to that of WT. At this stage, it is only possible to speculate that it is not required on the basis of the CA experiments shown in Figure 12, if it is assumed that the O2 protection is not totally due to the AcPyTACN anchoring. On the contrary, if such protection resulted only from the favourable orientation of the enzyme on the surface of the MWCNT, these preliminary results did not allow any further conclusions.

Size-exclusion chromatography to multi-angle laser light scattering with online refractive index (SEC-MALLS-RI).

Purified RrCODH D-cluster mutants and WT samples were injected manually into a SEC- which is available and used on Microsoft Excel. This tool is designed for semiquantitative analysis of iron-sulfur cluster formation/decomposition reactions over time, based on the fitting of the spectra from library of FeS clusters spectra.

Metal content determination and Methyl Viologen mediated CO oxidation activity in solution.

Metal content was determined thanks to colorimetric assay as previously described in Chapter 2. CO oxidation activity in solution was measured, as previously described in Chapter 2, in "standard conditions" in CO saturated 50 mM TrisHCl, pH 8.5, 12 mM

Methyl Viologen at 25 °C. RrCODH D-cluster mutants were suitably diluted in a range of concentrations from 10 to 500 nM monomer, in order to be able to measure accurate kinetics.

Electrochemical measurements.

The electrochemical experiments were carried out in a three-electrode electrochemical gas tight cell inside an anaerobic glove box (O2 < 2ppm, Jacomex) using a Biologic VMP3

Electrochemical analysis.

Γmax: the surface coverage is defined as the number of mole of enzymes immobilized for cm -2 of electrode surface and it is calculated according to:

Γ max = 𝑄 𝐹𝑛𝐴
Where Q is the charge expressed in Coulomb obtained from the integration of the oxidative peak, F is the Faraday constant expressed in C mol -1 (96485.34 C mol -1 ), n is the number of electrons involved in the redox Cred2→Cred1 system (2 electrons) and A is

Conclusion & Perspectives

The main purpose of this work was to bring the scientific community's attention back to Among all the future prospects that out results shed light, I would like to propose objectives that can be achieved in the near future. To this end, I would like to mention that in the context of the development of this project, Thomas Pichon has recently joined our group to start a PhD.

As briefly described in Chapter 1, Synthesis Gas or Syngas represents an economical and versatile substrate with widespread applications (Figure 1A). In the last decade, world production of syngas has almost tripled reaching about 900 GWth per year (Figure 1B) [START_REF] Advisors | Global Trends in Syngas[END_REF]. Actually, the ecologic problem of syngas production is due to the fact that its mainly produced from the gasification of fossil fuels, but it can alternatively be generated from biomass or other ecofriendly sources as shown in Table 1. Regardless the carbon source, the raw syngas is mainly composed by carbon monoxide. In fact, the H2/CO ratio is between 0.2 and 0.66 but for many applications a H2-enrichment processes is absolutely required [START_REF] Nanda | Advancements and confinements in hydrogen production technologies[END_REF]. However, while gasification and purification of gas from fossil raw materials is a wellestablished technology, relevant differences still exist when using biomass as feedstock and specific challenges still must be addressed. For example, the impurity levels in the gas are different in eco-derived gasses. The WGSR is the key reaction for the purification and upgrading of syngas. To date, WGSR is industrially performed and consists of two sequential stages: the first with a high temperature reactor at 350-500 °C with an ironbased catalyst (Fe2O -Cr2O3) and a second low temperature reactor at 200-220 °C with Cu-based catalyst (Cu-ZnO/Al2O3) [START_REF] Nanda | Advancements and confinements in hydrogen production technologies[END_REF]. In addition, the presence of contaminant species, such as sulfur or nitrate compounds in the inlet gas, even at concentrations as low as 100 ppm, can lead to catalyst poisoning and a decrease in the global process efficiency [START_REF] Ratnasamy | Water gas shift catalysis[END_REF]) (Alfano & Cavazza, 2018). Another important limitation of the reaction is the H2/CO ratio, which needs to be relatively high to avoid side reactions. To date, one challenge is the development of Power-to-syngas processes, allowing the production of syngas from water, CO2 and renewable energy via different possible ways. For example, the photo-electrolysis of water can be coupled with the reverse water gas shift reaction (RWGSR)(Figure 2) [START_REF] Daza | CO2 conversion by reverse water gas shift catalysis: comparison of catalysts{,} mechanisms and their consequences for CO2 conversion to liquid fuels[END_REF]. The optimization of WGSR, in its direct or reverse way, appears then as an attractive research topic in the field of catalysis for sustainable chemistry for the development of biomass, waste and CO2 valorization processes in order to close the carbon cycle. Ideally our goal will be to provide an enzymatic device to perform WGSR and RWGSR under thermodynamically favorable conditions, such as room temperature and atmospheric pressure with high selectivity avoiding side reactions. In other words, this means a reduced impact on climate change. Therefore, by observing the in vivo multiprotein complex that performs the biological WGSR, the feasibility of exploiting this multiprotein complex in vitro represents a fantastic challenge.

Thank then to the friends of Urbino for having been there in the first academic steps. Federica, Elisa, Valentina and Roberta, you are all fantastic women, what a stupid and stubborn teenager needs to grow up. Nikos, Kalvinos and coach Lazaros, to be good friends and for introducing me to Greek culture. Stevo, the best roommate with whom I have shared many experiences, good and bad! Dani, Carola and Laura for adventures in the unknown region, advice and "teachings".

So, thanks to all the Bolognese people! All my good friends: Ste, Mariano, Alessandro (Il bello, NODO and Skënderbej), Carlotta, Pusi, Marius and Lamberto. Serena and Maria, for accepting me for who I am and for helping me in my personal and academic growth and thousands other reasons to thank you. Then, a big thank you to the people at LBIC. Muso, for accepting a "turbulent and non-ordinary" student and teaching me to analyze a scientific article and introducing, step by step, to the in silico scientific research. Luca, you already know, but thanks again for having patience and trust in me in approaching the first manipulations in the laboratory. For solving all the problems, I have created, for sharing your method of doing research, for introducing me to the fascinating field of protein crystallography. And also, for being available in these years where I have continued to stress you from a distance. Val, to be strict but fair, your teachings have been fundamental over the years. Prof! for being my scientific father. You have been and are one of my main sources of inspiration, for giving me the opportunity and accompanying me in the first steps in research and for believing in me for this PhD project. The great esteem and admiration I have in you are the reasons why I want you to be part of the jury, to examine the results of these three years.

Finally, thanks to the people of Grenoble and Marseille. Lorenzo 2 , for being a true and sincere friend, from the first day we met and for all the bonheur spent together. Of course, thanks to Emm, for having patience with these two "bad guys". Thanks to my "unknown" neighbors, Marc and Giovanna, you are amazing people! Martin, for being like a brother from day one. Thank you for teaching me the tricks of the trade, for introducing me to boulder. Hind, for being the purest, most loyal and sincere person I know. The world needs people like you. Manel, being a friend to talk to about everything from funny to very sad things, for example how sick our society is. Bruno, for introducing me to the EPR and your professionalism. I really appreciated and felt honored to work with you. In addition, to giving me the possibility of meet interesting and nice people from the BIP and for making me discover Marseille! I would also like to thank all the CDD and CDI people I have met in these years at LCBM and BEA, thank you for accepting me even though I am not too sociable. I won't list the names, just to make sure I don't forget anyone. But thanks to BIOCAT and BEE people, in the end it is like I have been in the two équipe. I want also to thank some people in particular, Sandrine for being a source of scientific inspiration and for climbing, one of the strongest people I know. Clara and Gregory, for being there to help me even though I was always in a bad mood, at least you managed to make me smile in some moments of depression. Julien, thank you very much. For all the scientific advice and discussions, for your availability and for having helped me a lot over these three years, without you I would not have achieved these results and this level of knowledge and maturity. Marila, for having been my bridge between Italy and France, without you it would have been really hard, maybe now I wouldn't be here. Thanks to the "director" Stephane, the safety and the way of doing things are very inspiring.

Thanks also for being able to find a way to make a smile and your humanity, I have great respect in you.

Special thanks to my CSI committee, Carole, Vincent and Sandrine, for the constructive discussions that prompted me to look at my research and goals from different perspectives. Also, a great thank to LabEx ARCANE for the grant that allowed this thesis to happen.

I really thank the people who accepted to be part of the thesis committee: Dr. Carole Baffert, Prof. Luis M. Rubio, Dr. Carole Duboc and Prof. Stefano Ciurli. I am sure that we will have the opportunity to have a constructive and interesting scientific discussion. So, it is time to say thank you to the two people they have contributed mainly to the achievement of this important milestone, the supervisors. Alan, thank you for introducing me to the field of electrochemistry as well as nanotechnology, for patiently teaching me from the first day to yesterday! Thank you for being there to motivate me and find good results where I was seeing only strange things. But as you say: "I like strange things"! We both know it was tough at certain times, but with a lot of nonchalance you managed to deflect my complaints and motivate me to try again and again. Christine, for being the best PhD supervisor I know. This is because you worry not only about work, but also about my sanity. Without you all this would have been impossible, you met me that I spoke little English and I was quite disoriented outside Italy. But thanks to you, who, like a mother, accompanied me in the first steps, I was able to acquire a good confidence in myself and also a good level of English and France, which helped me a lot to integrate into society. I think it is not obvious to feel free to discuss personal problems without any filter with your supervisor and receive support in the worst moments. You have always been available, on weekends, at night or early in the morning, on holidays and other situations. Thanks for everything you have done as mt scientific mother. Good luck managing the BEE team and good luck to you, Thomas and all people involved in continuing this ambitious project.

CO2 metabolism plays a fundamental role in biology, as it represents a "building block" for the synthesis of organic molecules in living organisms. On the other hand, the excessive CO2 emissions due to human activity in recent decades requires today the search for effective solutions dedicated to the closure of the carbon cycle. Thus, it is interesting to develop some alternatives inspired by nature. In particular, carbon monoxide dehydrogenase (CODH) catalyzes the reversible reduction of CO2 to CO and represents an attractive biocatalyst in terms of activity, selectivity, ability to operate in mild conditions with low overpotentials. In addition, it does not require high production costs and uses earth abundant-metals. For these reasons, several studies have been conducted on [NiFe]-CODHs for applications in electrocatalytic CO2 reduction reaction (CO2RR).

[NiFe]-CODHs consist of a complex multimetallic NiFe4S4 active site, unique in biology, and additional FeS clusters involved in the electron transfer from the buried active site to the protein surface. Here, we present a novel and efficient method to overproduce the fully active recombinant [NiFe]-CODH from Rhodospirillum rubrum in the easy-to-handle bacteria E. coli. We also developed the stable and favorable immobilization of the enzyme on noncovalently functionalized carbon nanotubes (CNTs), subsequently integrated in a gas-diffusion electrode device This device has comparable performance to molecular and metal catalysts for CO2RR. To go further, the understanding of the intramolecular electron transfer within the enzyme has been undertaken, in order to optimize its capacity to work in direct electron transfer with the electrode. Other optimizations are considered, such as the direct evolution of the enzyme to provide a better resistance towards O2. All together, the results of this work have paved the way to study and use this powerful biocatalyst in the near future for biotechnological applications.

Le métabolisme du CO2 joue un rôle fondamental en biologie, car il représente un "élément de base" pour la synthèse de molécules organiques chez les organismes vivants. D'autre part, les émissions excessives de CO2 dues à l'activité humaine au cours des dernières décennies, nécessite aujourd'hui la recherche de solutions efficaces dédiées à la fermeture du cycle du carbone. Ainsi, il est intéressant de développer certaines alternatives inspirées de la nature. En particulier, la monoxyde de carbone deshydrogénase (CODH) catalyse la réduction réversible du CO2 en CO et représente un biocatalyseur intéressant en termes d'activité, de sélectivité et de capacité à fonctionner dans des conditions douces avec de faibles surtensions. En outre, sa production est économiquement intéressante et est basée sur l'utilisation des métaux abondants sur terre. Pour ces raisons, plusieurs études ont été menées sur les [NiFe]-CODH pour des applications dans des réactions de réduction du CO2 (CO2RR). Les [NiFe]-CODH sont constituées d'un site actif complexe multi-métallique NiFe4S4, unique en biologie, et de centres FeS additionnels impliqués dans le transfert d'électrons du site actif enfoui au sein de la protéine jusqu'à la surface de la protéine. Au cours de la thèse, nous avons développé une nouvelle méthode t efficace pour surproduire la [NiFe]-CODH recombinante active de Rhodospirillum rubrum chez la bactérie Escherichia coli, largement utilisé en biotechnologie. Nous avons également développé l'immobilisation stable et favorable de l'enzyme sur des nanotubes de carbone (CNTs) fonctionnalisés de manière non covalente, intégrés dans un dispositif d'électrode de diffusion de gaz. Ce dispositif possède des performances comparables à celles des catalyseurs moléculaires et métalliques pour le CO2RR. Pour aller plus loin, la compréhension du transfert d'électrons intramoléculaire au sein de l'enzyme a été entreprise, afin d'optimiser sa capacité à travailler en transfert direct d'électrons vers l'électrode. D'autres optimisations sont envisagées, comme l'évolution dirigée de l'enzyme vers une meilleure résistance à l'O2. Dans l'ensemble, les résultats de ce travail ont ouvert une nouvelle voie pour étudier et utiliser ce puissant biocatalyseur dans un avenir proche pour des applications biotechnologiques.

Figure 0

 0 Figure 0 ©Statistically Insignificant by Raf S.

  two centuries, the industrial revolution has led to the development and diffusion of new technologies to improve human life. Many of the discoveries have been and are still focused on energy demand, required for almost all economic sectors.

Figure 1

 1 Figure 1 Total Primary Energy Supply (TPES) by source, referred to World 1990-2018. The tons of oil equivalent or toe is a unit of energy defined as the amount of energy released by burning one ton of crude oil. It is approximately 42 GJ or 11.63 MWh. (Adapted from International Energy Agency, 2021)

Figure 2

 2 Figure 2 Comparison between energy development models and related CO2 emissions. Existing policies (Current Policies) and announced targets (Sustainable Development) slow the growth of global CO2 emissions until 2040 butare not strong enough to force a peak in an expanding energy system.[START_REF] Iea | Total primary energy supply (TPES) by source[END_REF]

Figure 3

 3 Figure 3 Net fluxes and transfers of carbon among the atmosphere, land, and water are depicted in this simplified representation of the carbon cycle. The diagram includes only fluxes of CO2 but no other carbon-containing greenhouse gases. These carbon flows include 1) emissions (red arrows); 2) uptake (black arrows); 3) lateral transfers (blue arrows); and 4) burial (blue arrows), which involves transfers of carbon from water to sediments and soils. Values are expressed in teragrams of carbon (TgC) per year. The increase in atmospheric carbon, denoted by a positive value, represents the net annual change resulting from the addition of carbon emissions minus net uptake of atmospheric carbon by ecosystems and coastal waters. The estimated increase in atmospheric carbon of +1,009 TgC per year. Asterisks indicate that there is 95% confidence that the actual value is within 10% (*****), 25% (****), 50% (***), 100% (**), or >100% (*) of the reported value. (Adapted from[START_REF] Birdsey | Second State of the Carbon Cycle Report (SOCCR2)[END_REF]

Figure 5

 5 Figure 5 Reaction steps for enzyme catalyzed CO2RR. (A) CO2 reduction to CO catalyzed by Carbon Monoxide Dehydrogenase and (B) CO2 reduction to CH3OH catalyzed by a three-step cascade of dehydrogenases.

Figure 6

 6 Figure 6 Schematic Calvin-Benson-Bassham cycle. Input and output molecules are highlighted in red. (Adapted from F.[START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]

Figure 7

 7 Figure 7 Schematic reductive Tricarboxylic Acid cycle. Input and output molecules are highlighted in red. (Adapted from F.[START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]

Figure 8

 8 Figure 8Schematic Wood-Ljungdahl pathway. Input and output molecules are highlighted in red. (Adapted from F.[START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]

Figure 9

 9 Figure 9 Schematic 3-hydroxypropionate bicycle. Input and output molecules are highlighted in red. (Adapted from F.[START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]

Figure 10

 10 Figure 10 Schematic 3-hydroxypropionate-4-hydroxybutyrate cycle. Input and output molecules are highlighted in red.(Adapted from F.[START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]

Figure 11

 11 Figure 11 Schematic dicarboxylate/4-hydroxybutyrate cycle. Input and output molecules are highlighted in red.(Adapted from F.[START_REF] Gong | Synthetic biology for CO2 fixation[END_REF]

Figure 12 (

 12 Figure 12 (A) Ribbon diagram of Oligotropha carboxydovorans [CuMo]-CODH (PDB code: 1N63) the overall structure is a dimer of heterotrimers. Right monomer is colored according to subunit with salmon for the CoxL subunit, gold for the CoxM subunit and cyan for the CoxS subunit. Cofactors in van der Waals representation. (B) Cofactors representation for the electron transfer. Distances are: 8.7 Å between FAD and the nearest [Fe2S2]-cluster, 12.4 Å between the two [Fe2S2]-clusters and 14.6 Å between the CuMo-MCD (molybdopterin cytosine dinucleotide) and its nearest [Fe2S2]-cluster (C) Active site is built from the bimetallic [CuSMo(=O)OH] cluster in which the two metals are bridged by a µ-sulfido ligand (µS). Cu is further coordinated by a thiolate of cysteine 388. While Mo is further coordinated with the dithiolate pyran ring of MCD, an hydroxo-group and an oxo-group. Molybdenum is green water, copper is brown, phosphor is orange, sulphur is yellow, nitrogen is blue, oxygen is red, and carbon is grey.

  the reversible oxidation of CO to CO2. The [NiFe]-or anaerobic-CODHs can be classified according to their biological role in monofunctional or bifunctional, the latter forming a complex with acetyl-CoA synthase (CODH/ACS). Based on the respiratory processes associated with CO oxidation, these anaerobic carboxydotrophs are classified in four groups: acetogens, methanogens, sulfate-reducers and hydrogenogens (Figure13).

Figure 13

 13 Figure 13 Scheme of anaerobic respirations, which can be coupled to CO oxidation. CODH for carbon monoxide dehydrogenase; arrow with e -for electron transport chain, some components of which are unknown.

Figure 14

 14 Figure 14 Scheme of Hydrogenogens CO metabolism. Biologic WGSR multiprotein complex CODH:CooF:Energy-Conserving Hydrogenase (ECH) for H2 evolution from CO oxidation and protons translocation across the cytoplasmatic membrane. Protons gradient usage by ATP-synthase to phosphorylate ADP to ATP.

Figure 15

 15 Figure 15 Rhodospirillum rubrum, TEM. Length: 4 microns (©Bergey's Manual Trust 1995)

Figure 16

 16 Figure 16 Simplified metabolic map of R. rubrum with CO and acetate as carbon source. The biologic WGSR multiprotein complex catalyze the H2 evolution from CO and led protons translocation across the cytoplasmatic membrane. Protons gradient usage by ATP-synthase to phosphorylate ADP to ATP. CO2 can then be fixed through the CBB cycle, the TCA, or the EM-CoA. R1,5P: ribulose-1,5-bisphosphate, 3PG: 3-phosphoglycerate, Ac-CoA: acetyl coenzyme A, aKG: a-ketoglutarate, AcAc-CoA: acetoacetyl-CoA, Cro-CoA: crotonyl-CoA, PHB: poly([R]-3hydroxybutyrate) (Modified from[START_REF] Revelles | Carbon roadmap from syngas to polyhydroxyalkanoates in Rhodospirillum rubrum[END_REF].

  . The advantage of the Ni-containing catalytic center is the flexible coordination geometry which allows the ability to catalyze a wide range of reactions. The protein environment is then fundamental and is directly linked to the catalytic properties. The latter are closely related to the nature of donor ligands involved in Ni coordination and solvent exposure. Frequently, the active site is in a buried pocket, where the surrounding protein environment protects it from oxidative damage. This feature requires dedicated channels for substrates/products diffusion, proton channels and electron relays that electro-connect the active site to the physiological partner(s), such as FeS clusters. Furthermore, the coordinating ligands drive the redox properties of the metals giving access to the different redox states involved during the catalysis, allowing the enzyme to catalyze the reactions as close as possible to the standard potential of the reaction. To satisfy these parameters, S-donor rich environments are favored in the redox enzymes, while for the non-redox enzymes the O/N-donor rich ligands are favored (Figure17)[START_REF] Alfano | Structure, function, and biosynthesis of nickel-dependent enzymes[END_REF].

Figure 17

 17 Figure 17 Active site of (A) Rhodospirillum rubrum CODH (1JQK) as an example for S-donor rich ligands and (B) Sporosarcina pasteurii Urease (4CEU) as an example for O/N-donor rich ligands, a distal water molecule (W3) was omitted because only the O-donors are shown.

  Rhodospirillum rubrum: CODH and Energy-Conserving Hydrogenase (ECH). The latter belongs to the [NiFe]-Hydrogenase family which represents one of the most ancestral biological systems for the energy metabolism of organisms. They depict an ancient link

Figure 19

 19 Figure 19 Schematic models of (A) R. rubrum ECH (cooMKLXUH) and Ferredoxin/CODH (cooFSCTJ) operons regulated via the downstream transcriptional activator CooA (cooA). (B) C. hydrogenoformans Z-2901 ECH (cooMKLXUHA2) and Ferredoxin/CODH (cooFS1) operons. (C) Schematic model of the CO-oxidizing:H2-forming enzyme complex from C. hydrogenoformans. Capital letters indicate the subunits of the enzyme complex. CO oxidation only at one active site of CO dehydrogenase is shown. FeS box indicate the presence of iron-sulphur clusters into the different subunits. The iron-sulphur clusters of the CooS1 and CooH are not shown. (Adapted from[START_REF] Hedderich | Energy-converting [NiFe] hydrogenases: More than just H2 activation[END_REF].

  : Enzymes found in classes I and II are called acetyl-CoA decarbonylase/synthase (ACDS). They are considered as ancestral CODH, found in archaea (mainly methanogens) and consist of five different subunits (αβγδε) that form oligomeric complexes where the alpha subunit displays the CODH activity. Class III CODHs are generally found in acetogens where CODH forms a bifunctional complex with acetyl-CoA synthase (CODH/ACS). The gene encoding ACS is generally contiguous to the one encoding [NiFe]-CODH, and ACS that is tightly associated with [NiFe]-CODH. Finally, class IV CODHs are monofunctional enzymes and their major physiological function is the oxidation of CO as for example, in the WGSR where CO is an energy substrate.

Figure 20

 20 Figure 20 Subunit composition of [NiFe]-CODHs. Monofunctional and bifunctional CODHs may be distinguished according to their subunit composition and physiological function. Multiprotein complexes are indicated by overlapping the forms of the corresponding subunits. Homology within classes is indicated by the same coloration.

(

  B-and B'-clusters) and a Ni-Fe-S cluster (C-and C'-clusters). Another [Fe4S4] cluster (Dcluster) connects the two α subunits. This organization of five iron sulfur clusters has also been observed in the other [NiFe]-CODHs classes (see below). Differently from the other [NiFe]-CODHs classes, the α2ε2 oligomer harbors two additional [Fe4S4] clusters (namely, E-, E'-, F-and F'-clusters).

Figure 21 (

 21 Figure 21 (A) Ribbon diagram of Methanosarcina barkeri α2ε2 [NiFe]-CODH component of the ACDS (PDB code: 3CF4). Side view of the α2ε2, the α-subunits coloured in cyan and salmon and the ε-subunits in purple and green. (B) Top view of the α2ε2 component C Side view of metal clusters in the α2ε2 complex.

Figure 22

 22 Figure 22 Ribbon diagram of Moorella thermoacetica α2β2 CODH/ACS (PDB code: 1OAO). (A) Overall structure of CODH/ACS, the two CODH subunits are salmon and cyan, the left ACS subunit is in the opened conformation, the right ACS subunit is in the closed conformation. The three domains of the ACS are green (first), gold (second) and purple (third). (B) Superimposition of the ACS subunit in opened and closed conformation, the latter is light gray.

  CO released by C-cluster induces the transfer of another CO into the next pocket to finally reach the A-cluster. This mechanism has been proposed based on the position of Xe atoms in the enzyme, with the Xe closest to A-cluster (Ni) at a distance of only 3.5 Å.

Figure 23

 23 Figure 23 Liquorice ribbon diagram of Moorella thermoacetica α2β2 CODH/ACS (PDB code: 2Z8Y). Location of xenon (numbered blue spheres) sites within the structure of the CODH/ACS complex. CODH β-subunits are shown in green and ACS α-subunits are in purple. The predicted hydrophobic CO tunnel pathway is highlighted with dots brush blue.(Adapted from[START_REF] Darnault | Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open α subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase[END_REF]

Figure 24

 24 Figure 24 Structures of the A-cluster of Moorella thermoacetica (A) from PDB code: 1OAO (B) from PDB code: 1MJG.The importance of Ni and the role of the C-cluster in CODH will be dealt exhaustively in the following section relating to class IV or monofunctional CODH.

Figure 25

 25 Figure 25 Ribbon diagrams of Rhodospirillum rubrum monofunctional CODH (PDB code: 1JQK). Homodimer (β2) contains two unique [Ni3Fe4S] clusters (C and C') and three [4Fe4S] clusters (B, B' and D). Relative clusters distance indicates in Angstrom. (B) monomer (β) domains composition: helical N-terminal in electric blue, central α/βRossmann-like is blue and C-terminal α/β Rossmann-like is light blue.

Figure 26 (

 26 Figure 26 (A) D-, B-and B'-cluster coordination by cysteine thiolates from the two helical N-terminal domains. (B) Structure of R. rubrum C-cluster. [Ni3Fe4S] cubane is coordinated by four cysteine thiolates: 338, 451, 481,531 and the Feu is coordinated by a cysteine 300 thiolate and histidine 265.

  [4Fe4S] cluster to a [3Fe4S] subcluster, the latter can bind Ni to form Ni•[Fe]-complex intermediate. This mechanism involves a bond breaking in the cluster to allow the insertion of the Ni atom. The Feu may facilitate the deprotonation of the thiol group of Cys531 which allows the mature Ccluster assembly.

Figure 27

 27 Figure 27Proposed model for the mechanism of Ni insertion into Ni-free CODH in R. rubrum (Adapted from[START_REF] Jeon | New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: Analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster[END_REF] An alternative mechanism has recently been proposed for the Ni insertion, based on crystallographic and spectroscopic studies[START_REF] Wittenborn | Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase[END_REF].Drennan et al., solved the structure of DvCODH where the C-cluster is fully oxidized. The structure revealed different organizations of the metallocluster (Figure28). The proposed model is in agreement with the "bond breaking" theory and the displacement of an iron atom and one sulfur atom. This is supported by the structure of C-cluster in an oxidized state, in which Ni, Feu and a sulfur are shifted, accompanied by conformational changes of different side chain residues, while the position of the remaining [3Fe3S] sub-cluster is unchanged. Ni occupies the Feu site present in the reduced C-cluster form and is coordinated by two cysteines (Cys302 and Cys519 in Dv) and one histidine (His266 in Dv).Cys519 that normally binds Ni in the reduced state adopts an alternative rotamer conformation. An additional Ni ligand is Lys556, which normally does not coordinate the reduced C-cluster. Feu and the sulfur ion also undergo changes in their coordination while remaining associated with the [3Fe-3S] partial cubane. Two additional Feu ligands are Cys302 and a conserved cysteine residue (Cys301) which is normally not involved in the coordination of C-cluster in the reduced state. The plasticity of these two conformations of C-cluster was demonstrated by the recovering of a canonical organization of C-cluster when oxidized CODH crystals were chemically reduced by sodium dithionite(Wittenborn et al., 2019b).

Figure 28

 28 Figure 28Proposed model for the conversion between oxidized and reduced state of C-cluster in DvCODH (PDB codes: 6B6V and 6B6Y). For example, the conversion from reduced to oxidized state is made possible by oxygen exposure, while the opposite is made possible by sodium dithionite (DTH) reduction. In the reduced state the atoms adopt the canonical conformation of C-cluster CODHs, while in the oxidized state the Ni, Feu and one of the sulfur are in a shifted position and the Cys301 Lys556 appears as new coordinating residues.In the same manner, the exposure of reduced CODH crystals to O2 led to a structure with the non-canonical oxidized C-cluster. The structure of the C301S mutant, which is catalytically inactive, and does not contain any Ni shows an intact [3Fe-4S] C-cluster core with a Feu ion that adopts two conformations: 70% of Feu is in its canonical position coordinated by His266 and Cys302, and 30% is in the canonical position of the Ni of full mature C-cluster (Figure29).

Figure 29

 29 Figure 29The DvCODH(C301S) C-cluster is a mixture of species. The C-cluster has been refined with an alternative conformation of Feu. At 70% occupancy, Feu is ligated by His266 and Cys302 in its canonical binding site. At 30% occupancy, Feu is incorporated into the Fe-S cubane portion of the cluster.

Figure 30 2

 302 Figure 30 Alternative proposed mechanism for Ni insertion into C-cluster. 1 The C-cluster is produced as [4Fe4S] scaffold. 2 The latter should be converted in a [3Fe4S]-Feu subcluster based on the "bond breaking" theory. 3 CooC may be involved in inducing a conformational change in the C-cluster in which the Feu becomes ligated by Cys301 (Dv). 4 CooCTJ may be involved in Ni insertion into the canonical Feu-binding site (as observed by crystallography). 5 Cluster reduction could then result in formation of the fully mature C-cluster (as observed by crystallography). (Adapted from Wittenborn et al., 2019b) These two model mechanisms raise several questions: is an open distorted [3Fe4S]-Feu subcluster a true intermediate of the maturation process? How can His265 (Rr) directly drive C-cluster assembly? Could it be spectroscopically and/or crystallographically possible to observe the proposed intermediate 2 and 3 shown in Figure 30?

Figure 31

 31 Figure 31 Representative operons encoding for CODH in: Rhodospirillum rubrum (Rr), Citrobacter amalonaticus Y19 (CaY19), Thermococcus sp. AM4 (Tc I and II for CODH I and II, respectively), Moorella thermoacetica (Mt), Desulfovibrio vulgaris Hildenborough (Dv) and Carboxydothermus hydrogenoformans (Ch I and II for CODH I and II, respectively).CODHs cooS genes and CODH maturation genes are highlighted with a cyan and yellow dot frames, respectively. Electron-conserving hydrogenase genes (cooMKLXUH) and Acteyl-CoA Synthase genes (acsBCD) are also reported. Ferredoxin (cooF) is orange, CO-dependent transcriptional activator (cooA) is purple.

Figure 32

 32 Figure 32Ni concentration dependence of photosynthetic and CO-dependent growth of R. rubrum strains. Cultures were spotted onto plates containing SAN medium supplemented with the indicated concentration of Ni and then incubated in the presence of CO either illuminated or in the dark. Δ followed by the Coo protein indicate the gene that were inactivated. (Adapted from[START_REF] Kerby | In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: Molecular and physiological characterization of cooCTJ[END_REF]

Figure 33 (

 33 Figure 33 (A) Ribbon diagram of Carboxydothermus hydrogenoformans CooC1 (PDB code: 3KJI) in complex with Zn and ADP. The monomers are cyan and salmon. The nucleotide binding domain (P-loop) is green. ADP molecules are depicted with van der Walls surface. (B) The metal binding site at the dimer interface. The Zn atom is grey which is coordinated by two cysteine residues from each monomer (Cys112 and Cys114).Thus, the presence of ADP seems to favor the metal binding. A model for the binding and release of nickel was proposed form these studies (Figure 34) (J. H. Jeoung et al., 2010): the Ni(II) binding induces the stabilization of the homodimer, in which the P-loop would be in an open conformation. The ATP binding promotes a conformational change that allows the release of the metal. The way in which this conformational change occurs is not clear and is probably related to the hydrolysis of ATP which can modulate the interaction of CooC with other protein partners (such as CODH). Hence, ChCooC1 will

Figure 34

 34 Figure 34 Proposed model for nickel binding and releasing for ChCooC1. The ADP-bound dimeric state (1) was described via X-ray crystallography (PDB code: 3KJG). While the Zn/ADP-bound ChCooC1 (PDB code: 3KJI) is used as a model for Ni/ADP-bound ChCooC1 (2a,2b and 3), because it is not possible to crystallize ChCooC1 with Ni. The possible ATP triggered conformational change is shown in the state (4) which allow the interaction with CODH and ATP hydrolysis dependent release of Ni[START_REF] Sun | Conjugated N4-Macrocyclic Cobalt Complex for Heterogeneous Electrocatalytic CO2 Reduction with High Activity[END_REF]. The cysteines in the CXC motif are represented in yellow, and the signature lysine is shown in blue. (Adapted from J. H.[START_REF] Jeoung | Crystal structure of the ATP-dependent maturation factor of Ni,Fe-containing carbon monoxide dehydrogenases[END_REF]

Figure 35

 35 Figure 35 Ribbon diagram of apo-CooT structures from Rhodospirillum rubrum (PDB code: 5N76) (A) and Carboxydothermus hydrogenoformans (PDB code: 6FAN) (B). The structures show the solvent exposed Ni(II) binding motifs: "Cys2" for RrCooT and "Cys2-His55" for ChCooT. The alignment of 91 non-redundant CooT sequences revealed the existence of three distinct nickel-binding motifs in the CooT family: the "Cys2-His55/56" motif, present in

  conserved "H-(W/F)-X2-H-X3-H" motif, suggesting a double function for CooJ both as a Ni chaperone and as a Ni storage protein. To confirm this dual role of CooJ, it has been shown that a C-terminal truncated version of CooJ with only 6 histidines and no cysteine

Figure 37 (

 37 Figure 37 (A) Ribbon diagram of the truncated Rhodospirillum rubrum CooJ (PDB code: 6HK5). Each monomer of the homodimer are in cyan and salmon, Ni(II) is depicted as a green sphere. (B) Structure of the Ni(II)-binding site. Ni(II) is coordinated by four histidines, two for each monomer. Additional two Ni ligands from a glutamate from a second homodimer (in gold) of the crystal. In solution these two ligands can be water molecules. (C) Diagram of the RrCooJ homodimer composed by the two coiled coil monomer (residues from 1 to 68) and the two independent flexible histidine tails (residues from 69 to 115). Each dimer bound one Ni(II) at the interface of the coiled coil monomer with a KD in nanomolar range. Additional four Ni(II) are bound two for each histidine tail with a KD in micromolar range.

  2014, Inoue et al. expressed and purified ChCODH-I, which unlike ChCODH-II possesses a CooC gene (cooC3) in the native operon (T.[START_REF] Inoue | Over-expression of carbon monoxide dehydrogenase-I with an accessory protein co-expression: A key enzyme for carbon dioxide reduction[END_REF]. The Histagged RecChCODH-I was produced in E. coli with a protocol based on that of Jeoung and Dobbek, with the only difference that the bacteria were grown aerobically before IPTG induction. Purified RecChCODH-I showed a specific activity of 2 270 U•mg -1 which is equivalent to 12% of the wild type enzyme activity. The co-production of the enzyme with its accessory protein CooC3 resulted in an increase of the specific activity to reach 8 060 U•mg -1 , corresponding to 43% of the native enzyme activity. Interestingly, both the RecChCODH-I and RecChCODH-I CooC3 enzymes showed a lower nickel content than the wild type enzyme, with values of 14 and 42%, respectively. The percentage of Ni in the recombinant enzymes coincides with the percentage of specific activity compared to the native ChCODH-I. The authors suggested that when CooC is in the same operon as CODH, its co-expression is necessary for Ni insertion into the recombinant CODH (T.

Figure 39

 39 Figure 39Organization of TcCODHs gene clusters. TcCODH-I gene cluster also contain cooF and cooC1. TcCODH-II also contain cooF(upstream), cooC2, cooT1, cooT2 and ECH genes (downstream). CooC3 is present in a gene cluster not related to CODHs gene. (Adapted from[START_REF] Benvenuti | The two CO-dehydrogenases of Thermococcus sp. AM4[END_REF] Another reason of their low CO oxidation activity could be that the role of CODHs would not be related to CO/CO2 interconversion. It would be rather related to other functions as proposed for ChCODH-IV which acts as a CO scavenger in defense against oxidative stress[START_REF] Domnik | CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2[END_REF]. Taken together, these results on the heterologous production of various CODHs allow us to hypothesize that there are different mechanisms for Ni insertion into CODHs and further studies are needed to elucidate these mechanisms.

Figure 40

 40 Figure 40 Structures of CODH C-clusters bound to (A) CO and water (PDB: 3CF4), (B) CN and water (PDB: 3I04), (C) n-BIC and water (PDB: 2YIV). Only the [Ni3Fe4S] cubane coordinated to the mononuclear iron (Feu) are shown. A ChCODH structure obtained at 1.36 Å resolution with Ni bound to the CN -without water molecule at Feu shows a CN -with a more linear geometry (Figure 41A). The CN competes with CO for the binding to Ni, as confirmed by the presence of cyanide in both ChCODH and MtCODH/ACS structures, and can also displace the water molecule in order

Figure 41

 41 Figure 41 Structures of CODH C-cluster bound to (A) CN (PDB: 3I39), (B) CO2 (PDB: 4UDX), (C) NCO -(PDB: 4UDY). Only the [Ni3Fe4S] cubane coordinated to the mononuclear iron (Feu) are shown.Based on spectroscopic investigations and chemical reasoning, the C-cluster exists in three different oxidation states: a fully oxidized and catalytically inactive Cox state, a one electron reduced Cred1 state which binds CO and a three electrons reduced Cred2 state which binds CO2[START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF]. At redox potentials above -200 mV, C-cluster is in the Cox state, which is diamagnetic (S = 0) and probably corresponds to a {[Ni 2+ Fe 3+ ]:[Fe3S4] 1 } state (J.[START_REF] Feng | Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum: Effect of Redox Potential on Catalysis[END_REF]. Below -200 mV, Cox is reduced by one electron and forms the paramagnetic Cred1 state (S = 1/2) which can be assigned as a {[ Ni 2+ Fe 2+ ]:[Fe3S4] 1-}

  Thus, C-cluster has several redox midpoints depending on its redox state: Cox/Cred1 ~ -200 mV and Cred1/Cred2 ~ -530 mV. B-clusters show a midpoint potential between -300 and -530 mV (Box/Bred ~ -418 mV), consistent with its role in electron transfer (J.[START_REF] Feng | Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum: Effect of Redox Potential on Catalysis[END_REF], while D-cluster adopts a diamagnetic state (S = 2) at potentials higher than -530 mV, which is coherent with the possible role in electron transfer[START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase[END_REF]. The g-values of several [NiFe]-CODHs, determined by Electron Paramagnetic Resonance (EPR), are shown in

Figure 43

 43 Figure 43 EPR and UV-visible spectra of R. rubrum [NiFe]-CODH. (A) Trace A, almost full oxidized FeS clusters. Trace B, 33 % of reduced FeS clusters, Cred1 state (g = 2.03, 1.88 and 1.71). Trace C, 66 % of reduced FeS clusters, Cred1 + Bred (g = 2.04, 1.97 and 1.88) states. Trace D, full reduced FeS clusters Cred2 (g = 1.97, 1.88 and 1.75) + Bred states. (Spangler et al., 1996) ( ̶ ̶̶ ̶ ̶ ̶̶ ̶ ̶̶ ̶ ̶̶ ̶ ̶̶ ̶ ) (B) full oxidized [4Fe4S] clusters show the typical absorbance from 350 to 480 nm. ( ̶ .. ̶ ) partially reduced [4Fe4S] clusters by incubation with 100 % CO for 35 minutes. ( ̶ ̶ ̶ ) full reduced [4Fe4S] clusters by Sodium Dithionite. The inset shows the difference spectrum for oxidized minus CO-reduced enzyme.[START_REF] Ensign | Nickel is required for the transfer of electrons from carbon monoxide to the iron-sulfur center(s) of carbon monoxide dehydrogenase from Rhodospirillum rubrum[END_REF].

  ): 0. Inactive state: when the cluster is fully oxidized (Cox state). A first reduction by one electron produces the catalytical ready Cred1 state.1. Cred1 → Cred1-CO: H2O binds to the Fe(II) ion and the CO binds to the Ni(II) in a bent geometry due to the steric hindrance of a conserved isoleucine.2. Fe-H2O deprotonation: this step determines the production of the nucleophilic hydroxide. Although binding to Fe should lower the pKa of the water molecule, residues surrounding the active site are expected to assist in the formation of nucleophilic hydroxide species. Candidates include a conserved lysine or histidine residues, but this role is not yet clear[START_REF] Kung | CHAPTER 7 One-Carbon Chemistry of Nickel-206 Containing Carbon Monoxide Dehydrogenase and Acetyl-CoA Synthase[END_REF].

3 .

 3 Nucleophilic attack: the deprotonation of the Fe-bound water to form hydroxide allows the CO to adopt a linear geometry, which would perfectly position it for the nucleophilic attack of the hydroxide to generate the Fe-Ni-bridging COOH species (carboxylic acid intermediate). 4. Fe-Ni bridging COOH deprotonation: a second deprotonation of the reaction intermediate, possibly involving the same residues mentioned above, produces the Ni-Fe bridging COO -(carboxylate intermediate).

→

  Cred1 state): two electrons are transferred to the B-cluster then the C-cluster goes back to its Cred1 state. The B-cluster then transfers the electrons either to D-cluster, or directly to an external electron acceptor such as the CooF ferredoxin in the biologic WGSR complex.

Figure 44

 44 Figure 44 Proposed catalytic and inhibitory mechanisms of the [NiFe]-CODHs C-cluster, consistent with crystal structures containing bound substrate and inhibitor molecules. (Adapted from Kung & Drennan, 2011) Electrons are transferred from C-cluster to the external acceptor via FeS clusters and vice versa. The access of gaseous substrates, protons and water from the protein surface to the buried active site (and vice versa) is made possible through specific gateways.

Figure 45 (

 45 Figure 45 (A) Gaseous substrates channel. Liquorice ribbon diagram of superimposed of MtCODH/ACS and ChCODH:n-BIC (PDB codes: 2Z8Y and 2YIV, respectively). Van der Waals representation of n-BIC molecules and Xe atoms. The Xe atoms numeration is the same proposed in Figure 23. Both together n-BIC molecules and Xe atoms show the gas channel from the C-cluster to the solvent which can be used by the CO/CO2 molecules during the catalysis. (B) Protons and water channels. Liquorice ribbon diagram of ChCODH (PDB code: 2YIV). H2O molecules are show as red spheres, portion of the non-structural water molecules are show as the solvent surface plus the structural water molecules inside the channel which connect the surface to the Feu. Polar channel for protons transport in and out of the active site, constituted of conserved histidine residues.

  Oxidoreductase such as CODH, Hydrogenase or Formate Dehydrogenase (FDH), are biocatalysts which during their reactions involve the production and transfer of electrons. As presented for the CODH, the activities are closely related to the electrical potential of their containing solution. These two common physical phenomena are the foundations of electrochemistry.Electrochemistry is branch of physical chemistry that studies the relationships between electric potential and chemical reactions involving a flow of electrons between two electrodes separated by a conductive electrolytic solution. Electrochemistry is therefore a technique that allows the study of redox active biomolecules. As shown in the previous sections, homogenous catalysis involves the transfer of electrons to or from an external partner, such as ferredoxin or methyl viologen. In electrochemistry the electrode, cathode or anode, plays the role of final donor or acceptor of these electrons. Redox biomolecules soon sparked interest in electrochemistry[START_REF] Heyrovsky | Polarographic studies with the dropping mercury kathode.-Part XIII.-The effect of Albumins[END_REF], and today more and more proteins are being studied by electrochemistry. This is because proteins can act as drugs, early-stage disease biomarkers, targets for drug development and discovery, or as selective catalysts.Redox proteins can be structurally diverse, from very small proteins (a single chain made up of hundreds of amino acids) to multiprotein complexes, such as mitochondrial complex I (NADH-Ubiquinone oxidoreductase) or photosystem I (plastocyaninferredoxin oxidoreductase). As shown above for the [NiFe]-CODH, the active site is frequently buried within the protein scaffold and the electron transfer proceeds via prosthetic cofactors (e.g., FeS clusters or FAD) from the active site to an external redox partner, such as ferredoxin or cytochromes. When the aim is to study these biocatalysts by electrochemistry, it is essential to partially reproduce the conditions in cellulo to allow the detection of electron flow. Thus, a first difference arises about the electric connection between the redox enzymes and the electrode, which will be discussed below, explaining the two main modes of electron transfer: mediated or direct.Achieving efficient electron transfer plays a critical role when the goal is to develop new energy conversion devices (such as enzymatic fuel cells and/or electroenzyme reactors) as well as enzymatic biosensors. The latter topic has been extensively studied in the past for medical applications. For instance, different commercial glucose biosensors are available, based on glucose oxidases[START_REF] Editor Newman | Home blood glucose biosensors : a commercial perspective[END_REF]. Furthermore the biosensors have been studied in order to detectDNA, bacteria, viruses, pollutants, etc.

Figure 47

 47 Figure 47 Schematic representation of the two mechanisms of electron transfer between electrode and redox enzyme/partner. (A) Mediated electron transfer (MET) by a redox partner or a small redox molecule (mediator). (B) Direct electron transfer (DET).To date, DET has been reported for many enzymes such as peroxidase (horseradish and cytochrome c peroxidases), laccase, bilirubin oxidase and dehydrogenase (cellobiose and fructose dehydrogenases, formate dehydrogenases)[START_REF] Do | Mathematical Modeling of a Porous Enzymatic Electrode with Direct Electron Transfer Mechanism[END_REF][START_REF] Andreu | Direct Electron Transfer Kinetics in Horseradish Peroxidase Electrocatalysis[END_REF](G.[START_REF] Gupta | Direct electron transfer catalyzed by bilirubin oxidase for air breathing gas-diffusion electrodes[END_REF][START_REF] Bollella | Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells[END_REF][START_REF] Miller | Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar-Driven Reduction of Carbon Dioxide[END_REF]. DET has also been

Figure 48

 48 Figure 48 Schematic representation of (A) physical adsorption and (B) cross-linking immobilization of enzyme at the surface of the electrode.Entrapment or encapsulation consists of compartmentalizing the enzyme in a polymer or in inorganic matrix, such as sol-gels (Figure49)[START_REF] Tielmann | Increasing the activity and enantioselectivity of lipases by sol-gel immobilization: further advancements of practical interest[END_REF])(R.[START_REF] Gupta | Entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects[END_REF]. The enzyme can be encapsulated in non-conductive polymers such as chitosan and cellulose (H.[START_REF] Liu | Polyphosphonate induced coacervation of chitosan: encapsulation of proteins/enzymes and their biosensing[END_REF][START_REF] Krajewska | Application of chitin-and chitosan-based materials for enzyme immobilizations: A review[END_REF], with the advantage of preventing direct contact of the enzyme with the environment. Furthermore, conductive polymers such as polypyrrole, polyaniline and polythiopene can be used for the immobilization of enzymes. The electrical wiring of the biocatalysts can be achieved through the modification of the polymer backbone with a redox entity acting as a redox mediator and ensuring charge transport diffusion has been extensively developed for enzyme immobilization and wiring. Polymers can also be produced in situ on the electrode surface by electrooxidation of the monomer which produces a matrix with the encapsulated enzyme.

Figure 49

 49 Figure 49 Schematic representation of enzyme encapsulation into conductive or non-conductive polymers, for its immobilization at the surface of the electrode.

Figure 50

 50 Figure 50 Schematic representation of enzyme immobilization with supramolecular interactions: (A) Biotin/Streptavidin, the latter represented as a cross, (B) Adamantane/Cyclodextrin functionalized enzyme and (C) NTA-Cu(II)-Histidine tagged enzyme.

Figure 51

 51 Figure 51 Schematic representation of enzyme covalent immobilization on the electrode surface: (A) amidic bound between the amine (lysine) and NHS esters, reactions of thiol (cysteine) with maleimide, reaction of nucleophilic residue with epoxide (B) click reaction between the azide functionalized enzyme and alkyne, Diels-Alder cycloaddition between.

Figure 52

 52 Figure 52 Schematic representation of single walled carbon nanotube (SWCNT) and multi walled carbon nanotube (MWCNT).

  combination of CNT and proteins is a prolific research field, as it has the potential to combine very different properties and surface chemistry of CNT compared to other supports. Several reviews report the uses of these hybrid systems (CNT-proteins) for example in the fields of sensing/detection[START_REF] Kwon | Conducting Nanomaterial Sensor Using Natural Receptors [Review-article[END_REF][START_REF] Holzinger | Synergetic Effects of Combined Nanomaterials for Biosensing Applications[END_REF][START_REF] Holzinger | Nanomaterials for biosensing applications: a review[END_REF][START_REF] Vashist | Advances in carbon nanotube based electrochemical sensors for bioanalytical applications[END_REF] and fuel cells[START_REF] Cosnier | Recent advances in carbon nanotube-based enzymatic fuel cells[END_REF][START_REF] Huang | Wearable biofuel cells based on the classification of enzyme for high power outputs and lifetimes[END_REF])(Le Goff et al., 2015). Two main types of CNT functionalization for the immobilization of different enzymes have been reported: non-covalent and covalent. Non-covalent attachment preserves the unique properties of both enzymes and CNT, but immobilized molecules can be gradually lost during the use of the CNTenzyme complex. Covalent conjugation provides lasting attachment but modify either the carbon sp 2 of the CNT or CNT defects. Non-covalent functionalization is interesting for the immobilization of biomolecules on CNT because it preserves the intrinsic properties of the CNT electrode and it is simple to implement.The simplest non-covalent functionalization proceeds through the adsorption of the molecule on the carbon nanotubes by a soaking step. This technique can be performed by direct physical adsorption on CNT (non-specific) or adsorption with the presence of auxiliary (specific) molecules such as polymers, surfactants and linking molecules. In the case of non-specific adsorption of enzymes, different interactions are involved, mainly hydrophobic ones. The hydrophobic region in the protein surface allows interaction with the sidewalls of the CNT, such as π-π stacking

  addition, the same work also shows that lysozyme interacts with nanotubes through protonated amino groups with CNT defect sites at pH values lower than the isoelectric point. At pH values higher than the isoelectric point, they interact through amine adsorption. For non-specific adsorption, the quantities of enzymes adsorbed on the CNT are influenced by various factors, such as the nature of the enzyme and the surface chemistry of the CNT. Different enzymes, for example soybean peroxidase, βglucosidase and chymotrypsin, can be adsorbed on CNT in large quantities of 575, 630 and 670 μg of protein per mg of CNT, respectively (Y.[START_REF] Gao | Covalent Immobilization of Proteins on Carbon Nanotubes Using the Cross-Linker 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-a Critical Assessment[END_REF][START_REF] Gómez | Immobilization of β-Glucosidase on carbon nanotubes[END_REF][START_REF] Karajanagi | Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes[END_REF]. Specific adsorption can be achieved via two main types of CNT functionalization: π-π interactions of aromatic molecules with the sidewalls of CNT and by coating with polymers.Molecules that contain polyaromatic groups or a π-extended network are able to functionalize the CNT by forming specific and directional π-π stacking interactions with the graphite surface of the nanotubes. This interaction might also improves the solubility of the CNT in the solvents[START_REF] Zhou | Non-covalent functionalization of carbon nanotubes for electrochemical biosensor development[END_REF]. Several compounds have been used for the immobilization of biomolecules on CNT for the development of electrochemical bioelectrodes. Among these, the aromatic groups are served by pyrene, naphthalene, and porphyrin derivatives (Figure53A).

Figure 53 (

 53 Figure 53 (A) Schematic representation of π-π stacking interactions of pyrene and naphthalene derivates and porphyrin with CNT (B) Schematic representation of enzyme immobilized on 1-Pyrenebutanoic acid, succinimidyl ester functionalized CNT.

  et al., 2013); 2[START_REF] Gentil | Oriented Immobilization of [NiFeSe] Hydrogenases on Covalently and Noncovalently Functionalized Carbon Nanotubes for H 2 /Air Enzymatic Fuel Cells[END_REF] 3 (Le Goff et al., 2010); 4[START_REF] Giroud | Anthracene-modified pyrenes immobilized on carbon nanotubes for direct electroreduction of O2 by laccase[END_REF]; 5[START_REF] Reuillard | Non-covalent functionalization of carbon nanotubes with boronic acids for the wiring of glycosylated redox enzymes in oxygen-reducing[END_REF]; 6[START_REF] Lalaoui | Hosting adamantane in the substrate pocket of laccase : direct bioelectrocatalytic reduction of O2 on functionalized carbon nanotubes Hosting adamantane in the substrate pocket of laccase : direct bioelectrocatalytic reduction of O 2 on functionalized car[END_REF] 78Bourourou et al., in 2013, succeeded in the functionalization of MWCNT with 1-(2anthraquinonylaminomethyl)pyrene and [bis(2-anthraquinonyl)-aminomethyl]-pyrene to immobilize and orientate laccase. These biocathodes were able to bioelectrochemically reduce oxygen, with an excellent stability over weeks.Holzinger et al., in 2011, achieved the multiple functionalization of SWCNT with three different molecules of pyrene derivatives: adamantane-pyrene, biotin-pyrene, and nitrilotriacetic acid (NTA)-pyrene. This rapid and reproducible procedure was carried out with nanotube-coated electrodes by means of a simple dip coating of the pyrene derivative molecules. The prepared multifunctional electrodes were then immobilized with cyclodextrin modified glucose oxidase, biotinylated glucose oxidase and histidine modified glucose oxidase, to finally develop a glucose biosensor. Adamantane-pyrene

  of [NiFe]-Hydrogenase from D. fructosovorans. The CNT/polymer bioelectrode displayed a MET-based electrocatalytic current density of 0.3 mA•cm -2 for H2 oxidation (Le[START_REF] Goff | Molecular engineering of the bio/nano-interface for enzymatic electrocatalysis in fuel cells[END_REF]. Other widely studied redox polymers are osmium-based hydrogels, based on a polymer backbone that is typically a polyvinyl chain and a redox coordination complex based on osmium (II) bearing groups such as imidazole or pyridine. The redox potential of osmium (III)/osmium (II) can be adjusted to cover the required redox potentials of the biocatalysts in order to enable efficient MET (Le[START_REF] Goff | Molecular engineering of the bio/nano-interface for enzymatic electrocatalysis in fuel cells[END_REF]. Minteer and colleagues modified polyethyleneimine with ferrocene for the immobilization of GOx and several dehydrogenases[START_REF] Aquino Neto | Membraneless enzymatic ethanol/O2 fuel cell: Transitioning from an airbreathing Pt-based cathode to a bilirubin oxidase-based biocathode[END_REF])(Aquino Neto, Hickey, et al., 2015)[START_REF] Knoche | Hybrid Glucose/O2 Biobattery and Supercapacitor Utilizing a Pseudocapacitive Dimethylferrocene Redox Polymer at the Bioanode[END_REF])(Aquino Neto, Milton, et al., 2015). It has recently been shown that a methyl viologen based hydrogel can protect oxygen-sensitive hydrogenases from high potential and oxidative deactivation[START_REF] Plumeré | A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage[END_REF][START_REF] Ruff | Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough under Oxidative Conditions[END_REF][START_REF] Oughli | A redox hydrogel protects the O2 -sensitive [FeFe]hydrogenase from Chlamydomonas reinhardtii from oxidative damage[END_REF].Unlike the non-covalent functionalization, covalent functionalization involves a chemical modification of the CNT, which induces a change in carbon hybridization from sp 2 to sp 3 , changing thus the native properties of the CNT[START_REF] Mallakpour | Surface functionalization of carbon nanotubes: fabrication and applications[END_REF].Moreover, as mentioned before, the CNT can already present some defects due to their production and dispersion in solution. Several covalent CNT functionalization techniques are available[START_REF] Singh | Organic functionalisation and characterisation of single-walled carbon nanotubes[END_REF][START_REF] Georgakilas | Organic functionalization of carbon nanotubes[END_REF](Hirsch, 2002), which can be divided in two main groups: the direct modification of the CNT sidewalls and the indirect modification of previous oxidized CNT, in order to direct the chemical modification on the O-containing groups generated by a first step of oxidation. A first oxidation of the CNT with reagents such as concentrated sulfuric acid, nitric acid and hydrogen peroxide allows the generation of O-containing groups such as carboxy, hydroxyl and epoxy groups or quinones. A commonly used technique is the immersion of the CNT in an oxidizing solution and together with a sonication bath, which allows CNT dispersion. These introduced defects may allow the immobilization of the enzyme through esterification and amide bond (Figure54).

Figure 54

 54 Figure 54 Scheme of indirect oxidized CNT functionalization for enzyme immobilization through esterification and amidation. Only carboxylic groups are represented, other O2-containing groups of oxidized CNT are: hydroxy, epoxy groups or quinones.

  [START_REF] Nazaruk | Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications[END_REF][START_REF] Goff | Facile and tunable functionalization of carbon nanotube electrodes with ferrocene by covalent coupling and π-stacking interactions and their relevance to glucose bio-sensing[END_REF].

Figure 55 (

 55 Figure 55 (A) Scheme of covalent functionalization of oxidized CNT with anthracene groups. (B) Simplified scheme of co-substrate (ABTS) or mediator functionalization of oxidized CNT (modified from[START_REF] Nazaruk | Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications[END_REF].

Figure 56 (

 56 Figure 56 (A) Direct covalent functionalization of CNT sidewall with carbene and nitrene. (B) Direct covalent functionalization of CNT sidewall with diazonium salts in order to expose hydrophobic/hydrophilic and electrostatic interactions or coupling with amino acids or sugar residues.

Figure 60

 60 Figure 60 Schematic representation of the H2/air enzymatic fuel cell. (Adapted from[START_REF] Gentil | Oriented Immobilization of [NiFeSe] Hydrogenases on Covalently and Noncovalently Functionalized Carbon Nanotubes for H 2 /Air Enzymatic Fuel Cells[END_REF] Another example of HFC using [NiFe]-hydrogenases and MCO was reported in 2016. GDE bioelectrodes showed a high current density of approximately 10 mA•cm -2 as steady state for both the oxidation of H2 and the reduction of O2. The integration of the GDE electrodes in a biofuel cell allowed the separation of the gas supply with consequent suppression of the oxidative inhibition of [NiFe]-hydrogenases. The HFC achieved a maximum power density of 8.4 mW•cm -2 at the cell voltage of 0.7 V under quiescent conditions[START_REF] So | Direct electron transfer-type dual gas diffusion H2/O2 biofuel cells[END_REF].

First, both

 both ChCODHs were shown to direct electrocatalyze the interconversion of CO2/CO. Thus, they investigated the potentials threshold of the known [NiFe]-CODH inhibitors: sulfide (Cox state), cyanide (Cred1 state), cyanate (Cred2 state) and O2 (Figure 61).

Figure 61

 61 Figure 61 Potential dependence of binding of inhibitors to ChCODH I. Red refers to the potential region over which the enzyme is inhibited, gray indicates no binding, and green indicates that binding leads to turnover. The dashed arrows indicate reactions that are slow compared to those indicated by full arrows. (Adapted from V. C.-C.Wang, Ragsdale, et al., 2013).Since cyanide (CN -) inhibits exclusively the Cred1 state, it acts as an inhibitor a potential below -300 mV. The addition of cyanide (1mM) into the electrochemical cell during the oxidation of CO at -280 and +80 mV resulted in both cases in the drop of current for the two ChCODHs. Cyanide inhibition was also tested under CO2 by its injection at more negative potential (-760 mV) during the second scan. The CO2 reduction current decreases at each scan due to the binding of cyanide in the Cred1 range of potentials.Chronoamperometry experiments were performed to evaluate the rate of inactivation and reactivation at low potential (release of CN -while Cred1 state is converted to Cred2 state) of cyanide inhibited ChCODHs. In figure62ABthe current is due to the respective oxidation of CO (-460 mV) and the reduction of CO2 (-560 mV). The injections of CN -were performed to reach a final concentration of 0.5 mM. These experiments were performed at different potentials to calculate the half-time inhibition, which is stable (all around 1-

Figure 63

 63 Figure 63 EPR spectra of ChCODH I (A) oxidized with benzyl viologen (B) oxidized with benzyl viologen and in presence of 1 mM or 20 mM (C) cyanate (D) reduced with CO (E) reduced with CO and in presence of 1 mM cyanate (F) reduced with dithionite (G) reduced with dithionite and in presence of 20 mM cyanate (A) 12 K, 41.2 mW, 9.38 GHz (B) 12 K, 41.18 mW, 9.38 GHz (C) 12 K, 41.2 mW, 9.383 GHz (D) 12 K, 41.22 mW, 9.382 GHz (E) 12 K, 41.21 mW, 9.379 GHz (F) 12 K, 41.21 mW, 9.38 GHz (G) 12 K, 41.21 mW, 9.381 GHz (V. C.-C.[START_REF] Wang | A unified electrocatalytic description of the action of inhibitors of nickel carbon monoxide dehydrogenase[END_REF] Finally, the inhibition of the Cox state by sulfide was also confirmed by electrochemistry (V. C.-C.Wang, Ragsdale, et al., 2013). Sulfide acts as an inhibitor at potential above -50 mV and at -260 mV is fast released due to full conversion of Cox to Cred1. O2 inhibition of ChCODH II was also investigated by chronoamperometry (Figure64) (V.C.-C.[START_REF] Wang | Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase[END_REF].The reaction with O2 was tested at +140 mV and -260 mV under 100 % CO. Before and after O2 injection, steps of prereduction were carried out a -760 mV. The results show that raising the potential from -260 to +140 mV protects the

Figure 64

 64 Figure 64 Effect on ChCODH II electrocatalysis of injecting O2-saturated buffer (0.5 mL) into the electrochemical cell (3 mL) under different conditions. The black line indicates times during which the electrode potential was poised at (A) +140 mV or (B) -260 mV, and red lines indicates times during which the electrode potential was poised at -760 mV for reductive reactivation. Experimental conditions: 25 °C, 0.2 M MES pH 7, 2500 rpm under 100 % CO atmosphere. Experimental conditions: 10 °C, 0.2 M MES pH 7, 2500 rpm, 100 % CO. (V. C.-C.[START_REF] Wang | Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase[END_REF] To better understand the irreversible inactivation of O2, similarly to experiments Figure64Bfurther experiments were conducted with a cyanide fully-inhibited ChCODH II. The injection of CN -before O2 resulted in a protection from O2 inhibition at -260 mV with a remaining electroactivity of approximately 50 % versus 1 % in Figure64B. Taken together, these results confirms that the O2 inhibition occurs from its binding to the Ni of the C-cluster, but that the decomposition of FeS clusters can also contribute, resulting in reduced DET as well as reduced electrocatalytic current. The oxygen inhibition of CODHs was further investigated by electrochemistry for DvCODH, ChCODH IV, TcCODH I and II, through similar approaches[START_REF] Merrouch | O2 Inhibition of Ni-Containing CO Dehydrogenase Is Partly Reversible[END_REF][START_REF] Domnik | CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2[END_REF][START_REF] Benvenuti | The two CO-dehydrogenases of Thermococcus sp. AM4[END_REF]. For the chronoamperometry experiments the potential was switched between -310 mV (catalysis and inhibition) and -560 mV (pre-reduction) and short transient exposition to a range of O2 concentration were tested for the different CODHs. From these first studies the DvCODH was shown to be more resistant to O2 compared to ChCODH II. For example, in the case of injection 1 µM of O2, the

Figure 66

 66 Figure 66 Schematic CO2 conversion with a dye-semiconductor-FDH photocatalyst system. Photoexcited electrons from the dye, RuP in (A) or DPP in (B), are transferred via the conduction band (CB) of TiO2 across the enzyme-material interface through the intraprotein [4Fe-4S] relays to the active site of FDH for the reduction of CO2 to formate. The oxidized dye is regenerated by triethanolamine (TEOA).[START_REF] Miller | Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar-Driven Reduction of Carbon Dioxide[END_REF]

Figure 67 (

 67 Figure 67 (A) Biological E. coli FHL-1 complex. FdhF, FDH; B/F/G, Fe-S cluster-containing proteins; HycE, [NiFe]-H2ase; HycD/C, membrane proteins. (B) FDH-ITO-H2ase nanoparticle (NP) system with enzymes immobilized onto ITO NP in solution. Species size not drawn to scale[START_REF] Sokol | Reversible and Selective Interconversion of Hydrogen and Carbon Dioxide into Formate by a Semiarti fi cial Formate Hydrogenlyase Mimic[END_REF]

Figure 68

 68 Figure 68 Schematic representation of the photoreduction of CO2 to CO system based on ChCODH-I and RuP.

  . To date, several non-biological catalysts have been combined with CNT-based electrodes, demonstrating CO2RR activity with high specificity towards aqueous CO/CO2 interconversion. Several cobalt-based compounds have been synthetized as: Cophtalocyanine (CoPc) or Co-quaterpyridine complexes (CoQpy) (Figure 69AB) (Kramer & McCrory, 2016)(X. Zhang et al., 2017)(Takanabe & Rodionov, 2016)(M. Wang et al., 2018).

Figure 69

 69 Figure 69 Structures of some metal-based molecular catalysts for CO2/CO interconversion: (A) Co-phtalocyanine (CoPc), (B) Co-quaterpyridine complex, (C) N4-macrocyclic Co complex (Co-CPY) (Adapted from Sun et al., 2020), (D) pyrene-substituted iron porphyrin (E) pyrene substituted [MnBr(2,2'-bipyridine)(CO)3].

■■

  proteins. The latter, were performed at the EPR facilities of the CNRS in Marseille under the supervision of Pr. Bruno Guigliarelli. We also performed a spectroscopic full characterization of the enzymatic properties such as pH and temperature dependence of the CO oxidation activity in solution, the pH and the temperature stability of the Rec-RrCODH and stability over time (91 % of remaining CO oxidation activity after at least 140 hours under anaerobic conditions at 21 °C). Insight into [NiFe]-CODHs metallochaperones dependent maturation. The differences between Rec-RrCODH and Rec-RrCODH CTJ in terms of specific activities in solution and MWCNT-immobilized, DTH-dependent redox activation in solution and Ni content strongly supports what was previously observed in vivo for the native enzymes (Kerby et al., 1997): the three metallo-chaperones CooC, CooT and CooJ are fundamental for the correct and full maturation of RrCODH. However, further studies are needed to fully understand the maturation mechanism of RecRrCODH and compare it already described mechanisms for other CODH. First determination of DET for RrCODH. Before the DET for the CODH from R. rubrum has never been shown. As for the other [NiFe]-CODHs, an efficient DET was obtained by immobilizing the Rec-RrCODH on a functionalized MWCNT electrode, as previously shown in the lab for other nickel enzymes such as the [NiFeSe]-Hydrogenase from D.

Finally, we were

  able to integrate the RecRrCODH functionalized MWCNT into a functional device. This first shows that a [NiFe]-CODH can catalyze the interconversion of CO2/CO at the gas/liquid interface. Secondly, the potential of [NiFe]-CODHs as robust biocatalysts for CO2RR was shown for the first time. This is because, unlike other previous electrochemical works on [NiFe]-CODHs, the GDE CODH-based electrode achieved electrochemical performances comparable to other catalysts for CO2/CO interconversion (See 2.3, Table

(

  10 µM concentration each): methylene blue, indigo carmine, phenosafranine, neutral red and methyl viologen. Redox potentials were measured with an Ag/AgCl/KCl (3 M) electrode. In the text, all potential values are given with respect to standard hydrogen electrode. After addition of a small amount of dithionite solution, potential stabilization was achieved in a few minutes, and sample was transferred into calibrated EPR tube and immediately frozen in the glove box in a cold ethanol bath. EPR experiments were performed on a Bruker ELEXSYS E500spectrometer equipped with an ER4102ST standard rectangular Bruker EPR cavity fitted to an Oxford Instruments ESR 900 helium flow cryostat. Spin intensity measurements were performed by double integration of EPR spectra recorded in non-saturating conditions and compared to a 1mM Cu(II)EDTA standard.

  wire was used ad counter electrode and the Saturated Calomel served as reference electrode. Potentials are referred to the Standard Hydrogen Electrode according to ESHE = ESCE + 0.242 V. Current densities are normalized towards the geometrical surface of the glassy carbon electrode or the gas-diffusion electrode (0.071 cm 2 ). The experiments were conducted at glove box temperature, 21 °C. The electrolyte was 50 mM TrisHCl buffer pH 8.5, 50 mM Bis-Tris propane pH5.5, 6.5, 7.5,

Figure S2 .Figure S3 .

 S2S3 Figure S2. Dithionite-dependent specific activity of Rec-RrCODH compared to Rec-RrCODH CTJ

Figure S4 .Figure S5 .

 S4S5 Figure S4. CVs of the Rec-RrCODH CTJ -functionalized MWCNT ADA electrode under Ar before (black) and after (blue) addition of 5 mM KOCN (50 mM TrisHCl, pH 8.5, v = 5 mV s -1 )

Figure S6 .

 S6 Figure S6. CVs of the Rec-RrCODH-functionalized (black) pristine MWCNT, (red) MWCNT ADA electrodes under Ar, CO and CO2 (50 mM Tris-HCl, pH 8.5, v = 5 mV s -1 )

Figure S8 .

 S8 Figure S8. pH dependence of the maximum current density measured at E = -0.8 V vs SHE by CV performed under CO2 (50 mM Bis-Tris propane pH5.5, 6.5, 7.5, 8.5 and 9.5, v = 5 mV s -1 )

Figure S10 .

 S10 Figure S10. (A) 3D image, (B) profile measurement performed by Laser Scanning microscopy and (C) Scanning Electron Microscopy image on a Rec-RrCODH CTJfunctionalized MWCNT ADA gas-diffusion bioelectrodes.

Figure S11 .

 S11 Figure S11. Electrocatalysis products analysed by Gas Chromatography. CO/CO2 production by of Rec-RrCODH CTJ -functionalized MWCNT ADA electrodes (A, B) and Gas Diffusion-Electrode (C, D). References: CO 100% (black) 0.59 min and CO2 (violet) 1.79 min (20 % CO2 80% N2). Aliquots of gas from the gas-tight electrochemical cell (10-50 µL) saturated with CO (A) or CO2 (B). Outgoing gas of gas diffusion electrode (10-50 µL) saturated with CO (C) or CO2 (D). Since a continuous flow of CO or CO2 was applied during the experiments, the saturated major peaks correspond to the substrates.

  have already successfully exploited the NTA-Ni to obtain a pure RecRrCODH preparation, thus our idea was to use the same technology to immobilize the RrCODH to the MWCNT-based electrode. Our strategy also involved the use of an alternative to the NTA ligand by studying the triazaciclonane-Ni complex at the electrode to provide increased stability towards enzyme immobilization. Another purpose of this study was to provide insights on the O2 resistance or inhibition of CODH from R. rubrum, based on the observed differences in terms of catalytic activities and O2 reactivity of the different monofunctional

  2+ and Fe 3+ . In biochemistry, commercial resins based on NTA-Ni and NTA-Co are widely used for the isolation of biomolecules with histidine tags. A pyrene derivate of the NTA-Cu has also been exploited for the immobilization of biomolecules on the electrode surface(Holzinger et al., 2011). Based on these observations, we were interested in confirming that the 1,4,7triazonane-1-carboxylic acid (TACN) moiety can also chelate different metals and retain its property for specific immobilization of the histidine-tagged RecRrCODH.The immobilization of RecRrCODH was tested on MWCNT-based electrodes functionalized with AcPyTACN-Co and AcPyTACN-Cu, furthermore for comparison, the immobilization was also tested with pyrene derivates of NTA-Ni, NTA-Co and NTA-Cu.The experimental procedures were performed as described above (Supporting information), except for the chelation of metal ions where 10 mM of Cobalt Sulphate orCopper Chloride was used. Cyclic voltammetry was used to characterize the electrocatalytic CO2 reduction activity for all types of bioelectrodes in a CO2 saturated buffer under quiescent conditions (FigureA1).

Figure A1

 A1 Figure A1 CVs of the RecRrCODH immobilized on AcPyTACN and NTA-pyrene MWCNT functionalized electrodes under argon (dotted) and CO2 (solid). Experimental conditions: 50 mM Tris-HCl, pH 8.5, v = 5 mV s -1 .

Furthermore, based on

  the fact that recombinant histidine-tagged proteins are isolated from the entire cell lysate using an NTA-Ni/Co resin, we tested the possibility of directly immobilizing the RecRrCODH on an AcPyTACN-Ni functionalized MWCNT electrode from the E. coli cell lysate. The extract was prepared as described in Chapter 2 (Supporting information), aliquots of 50 µL were incubated 4 hours with AcPyTACN-Ni functionalized MWCNT electrode. Then, the electrode was tested by cyclic voltammetry under argon and CO2 (Figure A2).

Figure

  Figure A2 (A) SDS/PAGE of pure 0.175 µg (2) and E. coli whole proteins extract(1). The amount of Rec-RrCODH in the whole proteins extract can be approximate to 0.2 mg•mL-1 , based on the same intensity of the pure. (B) CVs of. Experimental conditions: 50 mM Tris-HCl, pH 8.5, v = 5 mV s -1 .

  aldehyde groups and then by Schiff base condensation with the amino groups of histidines. From the original work, approximately 5-6 histidine residues were added to the surface of the protein after this chemical modification without affecting enzymatic performances. Therefore, without further characterization, we tried the immobilization of His-HRP on non-functionalized MWCNT or TACN/NTA functionalized electrodes. 20 µL of His-HRP at 2 mg•mL -1 were drop casted on the functionalized electrodes and incubated 4 hours at room temperature. The results of CVs under air or in the presence of 10 mM H2O2 are shown in Figure A3.

Figure A3

 A3 Figure A3 CVs of histidine added-HRP immobilized on different functionalized electrodes, under air (dotted) and in presence of 10 mM H2O2 (solid). Experimental conditions: 0.1 M PBS, pH 7.0, v = 10 mV s -1 .

■■■

  (2.4 mA•cm -2 ) functionalized MWCNT as compared to non-functionalized MWCNT(1.5 mA•cm-2 ). However, the current density of the TACN-Ni functionalized bioelectrode (0.6 mA•cm -2 ) is four-times lower as compared to NTA-Ni. This was not expected since this behavior was not observed in the case of RecRrCODH immobilized on TACN-Ni electrodes. However, the fact that histidines are located at multiple and nonspecific locations at the surface of HRP, on the contrary of CODH, might have a deleterious effect on the stable immobilization of HRP as well as its integrity at the surface of the electrode. To conclude these first experiments did not confirm the strong affinity of the TACN-Ni moiety for other histidine tagged proteins. Further experiments are needed to confirm this hypothesis with other engineered proteins. ■ Improved RecRrCODH orientation on the electrode surface. The ability to precisely control the orientation of the RecRrCODH on the surface of the MWCNT-based electrode was achieved by promoting selfassembly of the enzyme at the electrode surface via supramolecular interactions. Our results are in agreement with what was observed by L. Martin et al. and Spiccia et al. The electrocatalytic performances of the AcPyTACN-MWCNT bioelectrode exceed the previous reported for the CODH functionalized pyrene ADA -modified MWCNT electrode (Chapter 2). Original non-covalently functionalization of CNT for supramolecular enzyme immobilization. We provided an original MWCNT functionalization for histidine-tagged enzymes immobilization which provide enhanced electrocatalytic performances. However, in this first work it was not possible to further confirm the behavior of AcPyTACN-MWCNT electrode with other histidine tagged biomolecules such as His-HRP. At this point, it is possible to hypothesize that the strong affinity of the RecRrCODH is due to its homodimeric organization which provides two histidine tags, differently from the post-translational modified HRP. The synthesis of 1-acetato-4-(1pyrenil)-1,4,7-triazacyclononane and NMR characterization was performed by Mathieu Curtil (Plateau Synthèse Organique, PSO-DCM). Improved stability under aerobic conditions. One of the biggest bottlenecks for the development of nanodevices based on O2 sensitive biocatalysts is their instability under aerobic conditions. In particular, [NiFe]-CODHs, unlike Hydrogenases, are irreversibly inactivated for long periods of exposure to oxidative conditions, and can be slightly reactivated even in highly reductive conditions. Our results show that achieving accurate immobilization of biocatalysts can also provide better O2 tolerance. Insight into redox dependent O2 Inhibition of [NiFe]-CODHs. The dependent redox reactivity of [NiFe]-CODHs towards substrates and inhibitors has been investigated in the last decades. Regarding the reactivity of O2, it has been shown that Cred1 state is the most prone to O2-inhibition and Cox state can reduce its reactivity. Here, we showed that under catalytic conditions as well as in the Cred2 state the enzyme is slightly affected by O2 in the sense of CO2 reduction. However, further characterizations are needed to confirm the reactivity of O2 toward the Cred2 state. The role of D-cluster in [NiFe]-CODH from R. rubrum 4.1 -Introduction As discussed in the previous chapters, the two essential parameters required for the development of biocatalytic CO2RR nanodevices to take advantages of low overpotentials and high catalytic activity of enzymes are: I) an efficient DET between the active site of the enzyme and the electrode surface and II) a stability in time towards experimental conditions, such as temperature, pH or O2 for oxygen-sensitive enzymes.Although several electrochemical studies, have demonstrated that ChCODH-I and -II, DvCODH, the two TcCODHs and RrCODH are able to establish a DET, the precise characterization of the electron transfer pathway in these enzymes has never been clearly understood. In particular, it is not clear if the electrons pass from C-to B-cluster and then from B-to D-cluster or directly from B-cluster to the physiological partner (Figure1A). Indeed, the B-cluster is at a distance of 11.5 Å from the surface, which is compatible with a direct electron transfer without the requirement of the D-cluster. On the other hand, concerning their stability towards O2, it has been shown that O2 can inhibit the enzymes either via its direct binding to Ni atom, or via the oxidative damage of the FeS clusters impacting thus the electron relay. As show in Chapter 3, efficient anchoring of the RrCODH to the MWCNT can improve the catalytic performances but also the stability towards O2 inhibition. In support of this last result, the role of the Dcluster in O2 resistance has recently been demonstrated in DvCODH[START_REF] Wittenborn | Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase[END_REF][START_REF] Wittenborn | The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni-Fe Carbon Monoxide Dehydrogenase[END_REF]. It is worth mentioning that the D-cluster in DvCODH is made of a [2Fe2S] cluster instead of a [4Fe4S] cluster as observed in other described CODH (Figure1A). In CODH, the nature of the D-cluster depends on its binding motif: C-X2-C in the DvCODH sequence instead of C-X7-C in other CODHs (Figure1B).

Figure 1 (

 1 Figure 1 (A) Ribbon diagrams of monofunctional CODHs (PDB codes: Rr 1JQK, Dv 6B6V, Ch 6ELQ). Inset: relative clusters distances indicate in Angstrom. (B) CODHs amino acids sequences alignment and D-cluster motif binding. The prolonged exposure to air (2 days) of DvCODH crystals induces the formation of a catalytically inactive C-cluster in an oxidized conformation (as shown in Chapter 1 section 3.3.1) (Wittenborn et al., 2018).

Figure 2 (

 2 Figure 2 (A) Licorice diagram of superimposed CODHs: Rr is cyan (1JQK), Dv WT is orange (6B6Y) and C45G/T50C (6VWY). (B) [4Fe4S] D-cluster of Rr and Dv C45G/T50C CODHs coordination by Cys42 and Cys50 residues (Dv numeration).

Figure 3

 3 Figure 3 The [4Fe-4S] D-cluster in C45G/T50C Dv CODH crystals decays over time in air, whereas the B-and C-clusters remain intact. (A) 2Fo-Fc electron density at the D-cluster for crystals harvested immediately after removal from the anaerobic chamber; (B) 2Fo-Fc electron density for the D-cluster 2 hours or 2 days (C) after the crystal was removed from the anaerobic chamber; 2Fo-Fc electron density for the B-cluster (D) or C-cluster (E) 2 days after the crystal was removed from the anaerobic chamber.[START_REF] Wittenborn | The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni-Fe Carbon Monoxide Dehydrogenase[END_REF].

Figure 4

 4 Figure 4 Amino acids sequences of designed RrCODH D-cluster mutants.

Figure 5

 5 Figure 5 SEC-MALLS-RI of RrCODH D-cluster mutants and WT, 50 mM TrisHCl, pH 8.5, 150 mM NaCl (oxygenated buffer).

Figure 6

 6 Figure 6 SEC-MALLS-RI absorbance traces of WT (A) and C49T (B) RrCODHs incubated under air for different time scalesprior to be manually injected.

Figure 7

 7 Figure 7 Normalized UV-visible absorbance spectra of oxidized pure preparations of RrCODH WT and mutants.

Figure 8 (

 8 Figure 8 (A) UV-visible spectra of oxidized WT and mutants of RrCODH over time under air incubated, each spectrum was recorded every 15 minutes. (B) Fit-FeS v 1.1.1 spectra decomposition analysis. First 90 minutes (Up) under air time-dependent [4Fe4S] clusters decomposition (left) and [2Fe2S] clusters formation (right). And complete analysis until 390 minutes (below).

Figure 9

 9 Figure 9 CVs of the D-cluster mutants and WT RrCODH-functionalized MWCNT ADA electrodes under Argon at pH 8.5. Experimental conditions: 50 mM TrisHCl, pH 8.5, v = 5 mV s -1 .

  Figure10shows CVs of the mutant-functionalized MWCNT ADA under CO2 atmosphere. Almost all enzymatic preparations showed electrocatalytic current toward the CO2 reduction. Interestingly, for C49T and C41S, it was possible to observe a reversible catalysis, due to the oxidation of the CO produced from the CO2 reduction when the potential is closed to Cred2/ Cred1 one, showing that the enzymes are still able to catalyze the reaction in both directions.

Figure 10

 10 Figure 10 CVs of the D-cluster mutants RrCODH-functionalized MWCNT ADA electrodes under Argon (dotted) and CO2. Experimental conditions: 50 mM TrisHCl, pH 8.5, v = 5 mV s -1 .

Figure 11

 11 Figure 11 CVs of the WT and C49T D-cluster mutant RrCODH-functionalized MWCNT AcPyTACN electrode under Argon (dotted) and continuous flow of CO or CO2. Experimental conditions: 50 mM TrisHCl, pH 8.5, v = 5 mV s -1 , 1100 rpm.

Figure 12

 12 Figure 12 Chronoamperometry performed at Ep = -0.8 V vs. SHE in CO2 saturated 50 mM TrisHCl, pH 8.5, of C49T Dcluster mutant and WT RrCODH immobilized at AcPyTACN-modified MWCNT electrode after addition of O2. (WT are referred at Rec-RrCODH from Chapter 3).

  that the RecRrCODH precipitation conditions are around: 6-8 % PEG 8k, 0.1 M TrisHCl, pH 7.5, 1 mM DTT, 0.1-0.2 M CaCl2 and 4-8 % MPD. The protein crystals obtained from these last tests were cryoprotected in a mother liquor containing glycerol at 35 % and were flashcooled by quick immersion in liquid N2. Based on the recent results obtained on DvCODH suggesting that the [2Fe2S] D-cluster affords a better O2 stability to the enzyme, we designed the G44C/C49T mutant in order to convert the [4Fe4S] D-cluster to a [2Fe2S] one in RrCODH. However, in our hands, the results were very different from what was observed with DvCODH. Unexpectedly, the G44C/C49T mutations led to the loss of almost all FeS clusters, Ni and catalytic activities. Therefore, it is possible to conclude that a simple change in the sequence of the Dcluster binding motif from C-X7-C to C-X2-C as performed in reverse in DvCODH, is not possible in RrCODH.

Figure 13 (

 13 Figure 13 (A) Linear relation between Ni content of the dimer and specific activities of the RrCODH D-cluster mutants was derived from the specific activity and Ni content of the WT recombinant enzyme by using a simple equation: y = mx. Where y are the specific activities of CO oxidation in solution (■) or the CO2RR current densities of the immobilized enzymes (□), m is the slope (6567.5 U•mg -1 or 1,813 mA•cm -2 derived from 1.6 Ni/dimer : 10508 U•mg -1 /2.9 mA•cm -2 ) and x the Ni content of the dimer. (B) Normalized ratio of Ni content per Activities in solution (CO oxidation) and immobilized (CO2RR) of the WT and D-cluster mutants of RrCODH.

  MALLS system (Wyatt Dawn HELEOS-II 18-angle light scattering detector and Wyatt Optilab rEX refractive index monitor linked to a Shimadzu HPLC system comprising a LC-20AD pump and SPD20A UV/Vis. detector) using a Superdex 200 10/300 Increase size exclusion column eluted with buffer containing 50 mM TrisHCl, pH 8.5, 150 mM NaCl.An in-line refractive index detector (Optirex, Wyatt Instruments) was used to follow the differential refractive index relative to the solvent. The data analyzed using the ASTRA software v.6.1 and the molecular masses were calculated for each sample. Protein concentration in all samples was determined by integration of the differential refractive index (dRI) peak after injection of 20 µL protein. Oligomerization percentage contributes were calculated by integration of the dRI peaks.UV-visible spectroscopy.Oxidized enzymes were buffer exchanged to 50 mM TrisHCl pH 8.5 by using Micro Bio-Spin® 6 (Bio-Rad). The UV-visible spectra of oxidized RrCODH preparations were recorded on a UV-1800 spectrophotometer (Shimadzu Corporation) under anaerobic condition and aerobic conditions every 15 minutes for a total time of 390 minutes. The spectral decomposition of [4Fe4S] and [2Fe2S] was carried out by using Fit-Fes v.1.1.1

Potentiostat. 1 -

 1 pyrenebutyric acid adamantyl amide (ADA) and 1-acetate-4-(1-pyrenyl)-1,4,7-triazaciclononane (AcPyTACN) modified MWCNT electrodes (MWCNT ADA and MWCNT AcPyTACN ) were prepared as previously described in Chapter 2 and 3. The RrCODH immobilization on different functionalized MWCNT was carried out as previously described in Chapter 2, MWCNT-bioelectrodes were used as working electrodes. Pt wire was used as counter electrode and the Saturated Calomel served as reference electrode. Potentials are reported to the Standard Hydrogen Electrode according to ESHE = ESCE + 0.242 V. Current densities are normalized towards the geometrical surface of the glassy carbon electrode (0.071 cm 2 ). The experiments were conducted at glove box temperature, 21 °C. The electrolyte was 50 mM TrisHCl, pH 8.5. Chronoamperometry experiments were carried out as previously described in Chapter 2.

  an ancestral biocatalyst such as Carbon Monoxide Dehydrogenase. As previously described, also thanks to the catalytic efficiency of this ancient enzyme, during the early stage of Holocene it has promoted the development and spread of life on our planet as complex as we know it today. Geologists recently said that the Holocene is over and that we are actually living in the geological era of the Anthropocene. Although not widely endorsed by the geological community, this term indicates the beginning of the era when human socio-economic trends began to induce a dramatic change in the biological and geological balance of the Earth, known as Human-induced Climate Change. This is precisely in this regard that the need to reduce excessive CO2 emissions related to human activity motivated us to undertake this study. Obviously, with this work we do not intend to propose an alternative applicable today, but rather to help and facilitate the study of future viable ecological alternatives, based on what Nature selected long ago. Furthermore, we are not the first ones to try to show the potential of this robust biocatalyst. However, we hope that our contribution will help further work. Our protocol successfully achieved the easy access to highly active and stable R. rubrum [NiFe]-CODH allowing further biochemical, spectroscopic and structural characterizations. In addition, for the first time we have electrochemically shown the interest in using [NiFe]-CODH as a potential biocatalyst of CO2RR. Based on the same goal, the integration of RrCODH into a functional device, such as a Gas Diffusion Electrode, provides a proof of concept in development of concrete CODH-based nanodevices. Based on our recent findings, we have also begun to decipher the inherent drawbacks of [NiFe]-CODHs, such as low tolerance toward dioxygen and the role of metal cofactors, such as the D-cluster.

Figure 1 (

 1 Figure 1 (A) Synthesis Gas usage and final products of its conversion with relatively reactions. (B) World wide production of Syngas[START_REF] Advisors | Global Trends in Syngas[END_REF].

Figure 2

 2 Figure 2 Schematic representation of power-to-syngas process. Photocatalyst performs the electrolysis of water which is coupled to the reduction of atmospheric CO2 in the reverse WGSR (RWGSR) to obtain syngas.

Table 2

 2 Summary and comparison of the six natural carbon fixation pathways

	Pathway	Reaction			Energy inputs	Energy source(s)	Reference
							(Calvin & Benson,
	CBB cycle	3CO2	→	Glyceraldeide-3-Phospate	9 ATP, 6 NAD(P)H	Light, sulfur and H2	1948)(Sharkey, 2019)(Asplund-Samuelsson &
							Elton P, 2021)
	rTCA cycle	2CO2	→	Acetyl-CoA	2 ATP, 4 NAD(P)H	Light and sulfur	(M. C.

Table 3

 3 Chemolithotrophic growth on CO or H2/CO2 of aerobic carboxydotrophs. + CO and + H2/CO2 growth refer to the ability of the indicated cultures to grow in mineral media with CO or H2/CO2 as the sole carbon and energy source. + CBB refers to the potential for CO2 incorporation by the CBB cycle; s for slow; w for weak. (adapted from[START_REF] King | Distribution, diversity and ecology of aerobic COoxidizing bacteria[END_REF].

		Source	CO	H2 or CO2	CBB
	α-Proteobacteria		growth	growth	cycle
	Oligotropha carboxidovorans	Wastewater	+	+	+
	Pseudomonas thermocarboxydovorans Compost	+	-	+
	Pseudomonas carboxydohydrogena	Sewage	+	+	+
	Bradyrhizobium japonicum USDA 110	Rhizosphere	s	+	+
	Firmicutes
	Bacillus schlegelii	Freshwater sediment	+	+	+
	Actinobacteria
	Streptomyces thermoautotrophicus	Coal heap	+	+	?
	Mycobacterium smegmatis	Environment, human	+	+	+
	Mycobacterium gordonae	Human, water	+	+	?
	Mycobacterium tubercolosis H37Ra	Lung isolate	w	-	-
	Mycobacterium sp.JC1	Soil	+	-	+

Table 4

 4 Properties of several carboxydotrophs anaerobic

Table 5

 5 Metal content and specific CO oxidation activity of wild type CODH and variant of the C-cluster coordinating residues.

	CODH	Metal content (mol • mol CODH -1 )	Specific Activity	Reference
	variant	Ni	Fe	(µmol CO oxidized • min • mg CODH -1 )
	WT H265V	0.97 ± 0.09 0.24 ± 0.02	8.3 ± 0.9 7.9 ± 0.6	4,000 0.67	(Spangler et al., 1998)
	H265V+Ni	2.4 ± 0.1	8.1 ± 0.4	2.4
	WT	0.85 ± 0.05	8.97 ± 0.1	6,230 ± 74.9
	C338A C451A C451S	0.87 ± 0.06 0.92 ± 0.08 0.84 ± 0.09	7.29 ± 0.3 6.58 ± 0.7 7.59 ± 0.7	< 0.1 < 0.1 86.3 ± 7.5	(Jeon et al., 2005)
	C531A	0.92 ± 0.08	8.9 ± 0.1	6.9 ± 1.8
	These results established that the histidine and cysteine ligands of the C-cluster are
	critical for CO oxidation activity, although they are not fundamental for Ni insertion into
	the C-cluster. Concerning the iron content, the CODH variants show a slightly lower
	content but still in agreement with full metallated FeS clusters. The only ligand that
	affects the Ni content is His265. Interestingly, in vitro Ni(II) reconstituted H265V shows a

Table 6

 6 Comparison between R. rubrum CooC, Methanocaldococcus jannaschii UreG and Escherichia coli HypB.

Table 7

 7 Specific activity (U•mg -1 ) of CO oxidation and Nickel content of wild type and recombinant Carboxydothermus hydrogenoformans CODH-I and -II.

	ChCODH-II (No maturase in the operon)	Ni/dimer	U•mg -1 at 70°C	Reference
			and pH 8.0
	Wild Type	2	14,200	(Svetlitchnyi et al., 2001)
	Recombinant	N. D.	12,229

(J.-H.

Jeoung & Dobbek, 2007)

Table 8

 8 Specific activity (U•mg -1 ) of CO oxidation and Nickel content of recombinant Desulfovibrio vulgaris CODH.

	Sample	Ni/dimer	U•mg -1 at 37°C and pH 10.0
	DvCODH	0-0.5	< 5
	DvCODH+CooC	0.8-1.8	160
	DvCODH+CooC+Ni activation	N.D.	1 660

Table 9

 9 Specific

activity (U•mg -1 ) of CO oxidation and Nickel content of recombinant Thermococcus sp. AM4 CODHs.

Table 10

 10

[START_REF] Spangler | Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum[END_REF]

(Lindahl & Ragsdale,

Table 10

 10 Electron Paramagnetic Resonance (EPR) data for the CODHs from different sources

	Bacteria	B-cluster		C-cluster Cred1		C-cluster Cred2
		g-values		g-values		g-values
	R. rubrum a	2.04	1.93	1.89	2.03	1.88	1.71	1.97	1.87	1.75
	C. hydrogenoformans b	2.04	1.93	1.89	2.01	1.89	1.73	1.96	?	1.75
	M. thermoacetica c	2.04	1.94	1.9	2.01	1.89	1.65	1.97	1.87	1.75
	M. barkeri d	2.05	1.94	1.9	2.01	1.91	1.76	?	?	1.73
	D. vulgaris e	2.03	1.94	1.89	2.02	1.9	1.71	1.97	1.87	1.75

2MV 2+ → CO2 + 2H + + 2MV + CO2 reduction

  Scheme 1 CO-dependent reduction of oxidized methyl-viologen (MV) and CO2-dependent formation of carbonylhemoglobin (Hb-CO) from hemoglobin (Hb).

	CO oxidation	CO + H2O +

Table 11

 11 Specific activity of CO oxidation and CO2 reduction from several monofunctional CODHs. The pH buffers and temperatures are reported. Ch is for Carboxydothermus hydrogenoformans, Rr is for Rhodospirillum rubrum, Dv is for Desulfovibrio vulgaris, Tc is for Thermococcus sp. AM4; r.t. is for room temperature, n.s. is for not specified.

			CO oxidation		CO2 reduction
	CODH	pH T (°C)	U•mg -1	Ref.	pH	T (°C) U•mg -1	Ref.

Table 12

 12 List of pyrene-derivate compounds used for non-covalent functionalization of carbon nanotubes bioelectrodes and their applications.

Table 13

 13 summarizes the best electrocatalytic activities reported for mononuclear [NiFe]-CODHs, currently available. To date, no literature data are available on the direct or mediated electrocatalytic activities of CODH from Rhodospirillum rubrum, while for DvCODH and ChCODH IV, only mechanistic data are reported.

Table 13

 13 Best electrocatalytic mononuclear [NiFe]-CODHS activities for CO oxidation and CO2 reduction

	CODH	CO oxidation		CO2 reduction current	T (°C)	pH Ref
		I	E vs. SHE	Overp.	I	E vs. SHE	Overp.
		(mA•cm -2 )	(mV)	(mV)	(mA•cm -2 )	(mV)	(mV)
	Tc I	0.0027	-160	300	-0.0046	-760	300	37	6	1
	Tc II	0.03	-160	300	-0.048	-760	300	37	6	1
	Ch I	2.86	250	770	-0.4	-760	240	25	7	2
	Ch II	1.75	250	770	-0.16	-760	240	25	7

3 1 PFE on PGE electrodes (Benvenuti et al., 2020); 2 PFE on Polymyxin coated PGE electrodes (V. C.-C. Wang, Can, et al., 2013); 3 PFE on Polymyxin coated PGE electrodes (V. C.-C.

Wang, Ragsdale, et al., 2013)

Table 14

 14 Some products obtained from in vitro CO2 conversion with different enzymes (Modified fromÜnlü et al., 2021)

	Enzyme	Products Advantages	Disadvantages
	Formate	Formate	• Value-added product	• Forward reaction is favorable for
	Dehydrogenase			most FDHs
	(FDH)			• Requirement of improvement for
				long-term covalent immobilization
				• Expensive cofactor (NADH)
	Multiple	Methanol	• Value-added product	• Unstable for long-term activity
	Dehydrogenases			• Requirement of immobilization
	(FDH + FaldDH +			for industrial applications
	ADH)			• Expensive cofactor
	Carbon Dioxide	Formate	• No requirement of	• In cascade system, CO can easily
	Reductase		cofactor	inhibit the reaction
			• Reaction direction can
			easily drive by substrate
			concentration
	Remodeled	Methane	• No inhibition by H2	• Improvement required for
	Nitrogenase			turnover numbers and frequency
				• Oxygen-sensitive enzyme
	Pyruvate:Ferredoxin	Pyruvate		• Forward reaction is strongly
	Oxidoreductase			favorable
	(PFOR)
	Carbon Monoxide	Carbon	• CO easily convertible	• Oxygen-sensitive enzyme
	Dehydrogenase	Monoxide	to value-added products
	(CODH)
	Only two enzymes are capable of directly catalyzing the CO2 reduction: the CODH,
	already presented above, and the FDH.

Formate dehydrogenase catalyze the reversible oxidation of formate to CO2. Typically, FDHs require redox/co-substrate such as NAD(P) + /NAD(P)H.

Table 15

 15 Metal-based catalysts for aqueous CO2/CO interconversion.

		JCO	TOF		Overpot.	CO selectivity
	Catalyst	(mA•cm -2 )	(s -1 )	TON	(mV)	(%)	pH	Ref.
	CoPc2	165	3.9	ND	400	94	14	1
	CoPc	175	0.21	125	1910	95	14	2
	CoPc-P4VP	2	4.8	34,000	610	89	4.7	3
	CoFPc	4.4	1.6	ND	280	93	7.2	4
	CoPcCN/CNT	15	4.1	97,000	520	98	7.2	5
	CoQpy/CNT	19.9	12	89,905	440	99	7.3	6
	Co-COF	3.3	0.53	290,000	550	90	7	7
	CupolyPc/CNT	6.9	ND	ND	590	80	ND	8
	Au nanoneedles	22	ND	ND	240	95	ND	9
	Au nanowires	8.16	0.02	ND	ND	94	7.2	10
	Pd nanoparticles	9.76	0.16	ND	390	91	6.8	11
	Nanoporous Ag	8.7	0.002	ND	390	92	7.2	12
	FePhpyr/CNT	0.24	0.04	432	480	93	7.3	13
	Mn-bipy/CNT	5	0.04	2000	550	34	7.4	14
	*Mn

Table S1 .

 S1 State-of-the art catalysts for CO2 reduction

		Overpotential	Current	CO2	Catalyst	TOF	TON	Ref.
	Catalyst	(mV)	density	selectivity	Loadings	(s -1 )
			(mA cm -2 )	(%)	(nmol cm -2 )
	Rec-RrCODH CTJ /MWCNT ADA	180	4.2	100	0.052	420	800,000 This work
	Cobalt quaterpyridine/CNT	440	19.9	99	8.5	12	89,095	4
	CN-substituted phtalocyanin cobalt/CNT	520	15	98	18	4.1	97,000	5
	Cobalt phenanthroline/CNT	590	10.7	96	59	9.6	nd	5
	[MnBr(2,2′-bipyridine)(CO)3 on CNT	550	5	34	50	0.04	2,000	6
	Iron porphyrin on CNT	500	ca 5.5	90	6.4	0.05	750	7
	Poly-CuPc	300	6.9	80	-	-	-	8
	Gold nanoneedles	240	22	95	-	-	-

  Acetonitrile (HPLC) grade used for electrochemistry was obtained from VWR chemicals and used after drying on 4 Ȧ molecular sieves. NMR spectra were recorded on a Bruker AM 300 ( 1 H at 300 MHz, 13 C at 75 MHz) or a Bruker Avance 400 ( 1 H at 400 MHz, 13 C at 100 MHz). Chemical shifts are given relative to solvent residual peak. Mass spectra were recorded on a Bruker Esquire 3000 (ESI/Ion Trap) equipment.

	Electrochemical analysis. The electrochemical experiments in aqueous media were
	performed in 50 mM TrisHCl buffer pH 8.5 in a three-electrode electrochemical cell,
	using a Biologic VMP3 Multi Potentiostat, inside an anaerobic glove box (O2 <2 ppm,

  TrisHCl pH 8.5, 5 mM DTT, 5 mM DTH was diluted to 23 nM (monomer) in 50 mM TrisHCl pH 8.5, remaining 1 µM of DTT and DTH. Then, the enzyme was exposed to the air for several times (0-40 minutes) at 25 °C. The enzyme was further diluted to 5 nM (monomer) in anaerobic 50 mM TrisHCl pH 8.5, 5 mM DTT, 1 mM DTH and incubated 5 minutes in this buffer in order to pre-activate the enzyme before measuring the remaining specific activity.

	1 (411mg, 1.1 mmol) was then dissolved
	in acetonitrile (30 mL), sodium carbonate (3 g) and ethyl bromoacetate (252 µL, 2.3
	mmol) were added. The mixture was stirred at reflux for 6 h. Solvents were removed
	under reduced pressure. The crude product was dissolved in a 5M aqueous solution of
	NaOH (pH=12) extracted into chloroform (3 x 10 ml). The gathered extracts were dried
	over magnesium sulfate and the solvent removed under reduced pressure to give a
	viscous, yellow/orange oil. The product was purified by column CH2Cl2/Acetone affording
	a white powder (255mg, 63% yield). 1 H NMR (400 MHz, CDCl3), 𝛿(ppm): 1.14 and 1.25
	(3H, t, OEt, two resonance forms), 2.41 -3.41(12H, m, CH2 from macrocycle, two
	resonance forms), 3.27 and 3.39 (2H, s, NCH2COOEt, from two resonance forms), 4.05
	and 4.15 (2H, OEt, two resonance forms), 4.32 and 4.33 (2H, s, CH2-pyrene, from two
	resonance forms), 7.76-8.21 (9H, ms, CH from pyrene), 8.48 and 8.50 (1H, s, NCHO, from
	two resonance forms). 13 C NMR ( 100 MHz, CDCl3) 𝛿(ppm): 14.18 and 14.32 (CH3), 46.72
	(CH2), 47.43 (CH2), 50.14(CH2), 51.12(CH2), 53.84(CH2), 53.94(CH2), 54.60(CH2),
	54.80(CH2), 54.88(CH2), 55.56(CH2), 57.94(CH2), 58.08(CH2), 58.39(CH2), 58.47(CH2),
	60.37(CH2), 60.43(CH2), 61.18(CH2), 61,71(CH2), 76,83(CH2), 77.09(CH2), 77.34(CH2),
	124.04 (CH) 124.40 (CH), 124.42 (CH), 124.45 (CH), 124.76 (Cq), 124.82 (Cq), 125.01 (Cq),
	125.08(CH), 125.10(CH), 125.18(CH), 125.33(CH), 125.91(CH), 126.01(CH), 127.09(CH),
	127.16(CH), 127.29(CH), 127.40(CH), 127.49(CH), 128.13(CH), 128.31(CH), 129.81(Cq),
	129.87(Cq), 130.88(Cq), 130.89(Cq), 130.93(Cq), 131.28(Cq), 131,33(Cq), 132.74(Cq),
	133.16(Cq), 163.84 (CH), 171.78 (Cq), 172.10 (Cq)

Table 1 .

 1 Fitting parameters for the Langmuir-Freundlich isotherm model from figure

	4A.
		%Lossmax	KImid app (L mol -1 ) n	R2
	Rec-RrCODH	94 (+/-12)	4 (+/-) 1	0.5	0.995
	His-tagged Rec-	43 (+/-2)	12 (+/-3)	0.5	0.999
	RrCODH

Table A1

 A1 Maximum current density for CO2 reduction at -0.75 V vs. SHE achieved by RecRrCODH immobilized on different functionalized MWCNT electrodes.

	MWCNT Functionalization I / mA•cm -2
	NTA-Ni	2.82
	TACN-Ni	2.93
	NTA-Co	2.45
	TACN-Co	2.61
	NTA-Cu	2.76
	TACN-Cu	3.38

Table 1

 1 Oligomerization

	RrCODH		Tetramer/Trimer	Homodimer	Monomer
			(%)	(%)	(%)
	WT		0.0	100.0	0.0
	C41S		0.2	40.0	59.8
	C49S		0.0	97.9	2.1
	C49T		0.0	100.0	0.0
	C41A/C49S		1.2	71.3	27.5
	C41S/C49S		1.1	87.9	10.9
	aG44C/C49T		ND	ND	ND
	anG44C/C49T	ND	ND	ND
	aG44C/C49T	ΔiscR	0.0	78	12

percentage of RrCODH D-cluster mutants and WT calculated by integration of the dRI peaks from the results reported in Figure

5

. The low resolution of aG44C/C49T and anG44C/C49T does not allow the calculations.

Table 2

 2 RrCODH WT and C49T oligomerization incubated under air at different time scale. Percentage calculations refers to time 0 hour.

Table 3

 3 RrCODH

	Enzyme		A420/A280	[4Fe4S] (%)	Fe/Dimer
	WT		0.38 ± 0.01	100	19.0 ± 1.0
	C49S		0.21 ± 0.05	55 ± 13	9.4 ± 1.5
	C49T		0.27 ± 0.04	75 ± 8	13.5 ± 2.5
	C41S		0.23	60	11.2 ± 1.8
	C41A/C49S		ND	ND	6.5 ± 1.7
	C41S/C49S		0.28 ± 0.02	74 ± 5	14.2 ± 2.7
	aG44C/C49T		ND	ND	4 ± 1
	anG44C/C49T	ND	ND	9.4 ± 1.11
	aG44C/C49T	ΔiscR	ND	ND	9.4 ± 1.12

mutants purity coefficient (A420/A280), percentage of [4Fe4S] relative to native RrCODH and Fe(II) dosage. . The values with error are referred to at least 2 different preparations

Table 4

 4 Methyl Viologen-Mediated CO oxidation activity in solution and metals content of WT and D-cluster mutants of RrCODH. Theoretical specific activities are calculated relative to the WT Ni amount per dimer and specific activity. of the WT enzyme in the same experimental conditions. To further characterize their catalytic properties, these enzymatic preparations were studied electrochemically. RrCODH D-cluster mutants were immobilized on 1-pyrenebutyric acid adamantyl amide (ADA) modified MWCNT electrodes (MWCNT ADA ). All enzymes preparations were diluted to 38 µM monomer and incubated 4 hours with MWCNT ADA

	Enzyme	Fe/Dimer Ni/Dimer Specific Activity (U•mg -1 )
					Measured	Theoretical
	WT 1		19.0 ± 1.0 1.6 ± 0.2	10,508	10,508
	C49S		9.4 ± 1.5	0.4 ± 0.2	6.2 ± 5	2,627
	C49T		13.5 ± 2.5 1.2 ± 0.1 4,271 ± 220	7,881
	C41S		11.2 ± 1.8 0.3 ± 0.1	514 ± 35	1,970
	C41A/C49S		6.5 ± 1.7	0.2 ± 0.1	368 ± 17	1,313
	C41S/C49S		14.2 ± 2.7 1.0 ± 0.2	7.8 ± 2	6,567
	aG44C/C49T		4 ± 1	0.2 ± 0.1	< 1	1,313
	anG44C/C49T	9.4 ± 1.11 0.2 ± 0.1	< 1	1,313
	aG44C/C49T	ΔiscR	9.4 ± 1.12 0.4 ± 0.2	1.7± 0.5	2,627

Table 5

 5 Surface coverage Γmax (pmol•cm -2 ), potentials of oxidation, reduction and half wave potentials (E1/2) of the Cred1/Cred2 redox system.

	Enzyme	Γmax (pmol•cm -2 )	Oxidation	Potential (V vs. SHE) Reduction	E1/2
	WT (Chapter 2)	52 ± 2			-0.59
	WT		60	-0.54	-0.65	-0.59
	C49S		24	-0.45	-0.60	-0.52
	C49T		38	-0.54	-0.61	-0.57
	C41S		13	-0.49	-0.61	-0.55
	C41A/C49S		3.2	-0.52	-0.58	-0.55
	C41S/C49S		1.5	ND	-0.48	ND
	aG44C/C49T	ΔiscR

Table 6

 6 Electrocatalytic properties and specific activities comparison between in solution and immobilized WT and Dcluster mutants of RrCODH.

	noteworthy that, despite having negligible activity in solution the aG44C/C49T ΔiscR mutant
	still exhibit electrocatalytic current density of 0.28 mA•cm -2 towards CO2RR, although
	the efficiency of DET seems compromised as confirmed by lower current densities and
	TOF compared to WT enzyme, which is in agreement with the activities measured in
	solution. The functionalized C49T-MWCNT AcPyTACN electrode was also tested in CA
	experiment under CO2 saturated condition with injection of O2 saturated (Figure 12). As
	demonstrated for the WT enzyme, a favorable immobilization of the RrCODH on the
	MWCNT surface could also improve oxygen deactivation under CO2 catalysis (Chapter
	3). The same type of experiment was performed for C49T which showed a similar result,
	allowing to suggest that AcPyTACN anchor could improve the O2 tolerance of the C-
	cluster, even in the absence of the D-cluster.

From these results we can conclude that all D-cluster mutants achieved DET once immobilized on functionalized MWCNT. Furthermore, electrocatalytic currents for each mutant follow the same trend observed with the activities measured in solution. It is

Table 7

 7 Crystallization conditions tested for RecRrCODH at 25 mg•mL-1 , mixed 50/50 with the crystallization solution.

0.1

M TrisHCl, pH 7.5, 1 mM DTT, 0.4 M CaCl2 PEG 8k 6 % 10

		%	14 %	18 %	22 %
	A1	A2	A3	A4	A5
			2.5 % MPD
	B1	B2	B3	B4	B5
			5 % MPD
	C1	C2	C3	C4	C5
			7.5 % MPD

Table 8

 8 Crystallization conditions tested for RecRrCODH at 25 mg•mL -1 , mixed 50/50 with the crystallization solution.

0.

1 M TrisHCl, pH 7.5, 1 mM DTT

	6 %	8 %	10 %	12 %	14 % PEG 8k
			7.5 % MPD
	A1	A2	A3	A4	A5
			0.2 M CaCl2
	B1	B2	B3	B4	B5
			0.3M CaCl2
	C1	C2	C3	C4	C5
			0.4 M CaCl2

Table 10

 10 Crystallization conditions tested for RecRrCODH at 25 and 17.5 mg•mL-1 , mixed 50/50 with the crystallization solution.

Table 11

 11 Crystallization conditions tested for RecRrCODH at 25 mg•mL -1 , mixed 50/50 with the crystallization solution.

Table 8

 8 Yield of pure preparation of RrCODH D-cluster mutants.

	RrCODH:		Pure enzyme (mg•L -1 )
	C49S		5.5 ± 0.4
	C49T		4.8 ± 0.2
	C41S		3.1
	C41A/C49S		~ 5.1
	C41S/C49S		4.7
	aG44C/C49T		~ 7.1
	anG44C/C49T	~ 0.8
	aG44C/C49T	ΔiscR	~ 9.7

Table 1

 1 Composition of synthesis gas derived from various carbon sources (Adapted fromAlfano & Cavazza, 2018)

		Composition (vol %)
	Source	H2	CO	H2/CO CO2	N2	CH4 Other
	Coal gasification	29.4 59.4 0.49	10	0.6	-	0.6
	Charcoal	29	48	0.60	-	-	48	-
	Grass straw	2.6	12.9 0.20	17.4 64.2 2.1 0.8
	Demolition wood/paper residue 6.1	9.2	0.66	16.1 63.2 2.8 2.6
	Biomass (Updraft gasifier)	11	24	0.46	9	53	3	-

(Chapter 1) As expected, aG44C/C49T and anG44C/C49T did not show any measurable activity, supported by their global instability. Surprisingly, the other RrCODHs preparations retained partial CO oxidation activity. However, the activities are lower than the theoretical expected, calculated from the linear correlation between the amount of Ni

Acknowledgments

Here we are, the acknowledgments section. After these three years, I can understand what it means to do a PhD. Reaching this aim was/is challenging. It was a phase of growth from a scientific point of view, as now I feel more confident in myself and in the contribution, I can make to scientific research. Furthermore, this project has given me the opportunity to be more aware of the Human-Earth balance and how to try to contribute to the improvement of our society. In addition, it was also a time of personal growth, as this experience feels like the last stage of adolescence and the beginning of adulthood. I decided to put the acknowledgments here at the end, because I would like to thanks to all the people who allowed me to be here today to finish writing this thesis. I will follow a chronological order, as I don't want any of the following people to think they are more or less important than the others. Personally, I do not like classify/rank people, for me we are all the same. This is for you Gemma. Then, I thank my family and my parents, uncles Gennaro, Lucia and Emmanuela. Francesco, for having financed my first academic studies, you are a real and good brother. Mom, to always be there to think of me, to support me, to listen me complain (always), to advise me, for the patience and strength you had with me. Dad, for giving me an optical microscope and letting me "play" since my childhood in your laboratory. This has allowed me since I was a child to be attracted to Nature and its complex mechanisms. Lino and Andrea, I don't need to use words for you, just thank you for being part of my life and for being my non-biological brothers. Thank to my non-biological sister, Stella. I met you when I still did not speak "correct" Italian, thanks for being my personal Italian teacher, for introducing me to and strengthening my interest in Art! "Otherwise, Earth without Art is only Eh"

5 minutes) until very reductive potentials (-760 mV) (V. C.-C. Wang, Ragsdale, et al., 2013). Wang, Ragsdale, et al., 2013) Concerning the reactivation tests (Figure 62CD), a high potential was previously applied for 10 minutes in the presence of 2 mM CN -, in order to completely inhibit the enzyme.

The authors stated that, up to -560 mV, there was no reactivation, while, at -760 mV, the half-time reactivation was 19 seconds for ChCODH I and spontaneous for ChCODH II (V. C.-C. Wang, Ragsdale, et al., 2013). On the other hand, cyanate (NCO -) inhibiting exclusively the Cred2 state, exhibits the opposite behavior of cyanide by binding and inhibiting the enzyme at potentials below -500 mV and being released at high potentials.

The addition of 6.67 mM NCO -was tested at -760 mV under either 100 % CO2 and a mixture of 50 % CO2 and 50 % CO. The injection of cyanate resulted in a rapid full inhibition of CO2 reduction while the inhibition of CO oxidation was negligible under 100 % CO2 and 50 % CO2 and 50 % CO, respectively. The exclusive binding of cyanate to the Cred2 state was also confirmed by EPR.

UV-visible spectra of oxidized CODH

Protein samples were dialyzed in two successive baths of 500 mL of 50 mM TrisHCl pH 8.5 in an anaerobic glove box (O2 <2 ppm, Jacomex). The UV-visible spectra of oxidized Rec-RrCODH and Rec-RrCODH CTJ were recorded on a UV-1800 spectrophotometer (Shimadzu Corporation).

Size-exclusion chromatography coupled to multi-angle laser light scattering with online refractive index (SEC-MALLS-RI)

Purified Rec-RrCODH and Rec-RrCODH CTJ samples were injected manually into a SEC-MALLS system (Wyatt Dawn HELEOS-II 18-angle light scattering detector and Wyatt

Optilab rEX refractive index monitor linked to a Shimadzu HPLC system comprising a LC-20AD pump and SPD20A UV/Vis detector) using a Superdex 200 10/300 Increase size exclusion column eluted with buffer containing 50 mM TrisHCl, pH 8.5, 150 mM NaCl, 1 mM TCEP. An in-line refractive index detector (Optirex, Wyatt Instruments) was used to follow the differential refractive index relative to the solvent. The data were analyzed using the ASTRA software (version 6) and the molecular masses were Umberto Contaldo, [a,b] Mathieu Curtil, [a] Christine Cavazza,* [b] and Alan Le Goff* [a] [a] U. Contaldo, M. Curtil, Dr A. Le Goff Univ. Grenoble Alpes, DCM UMR 5250, BIOCEN, F-38000, Grenoble, France E-mail: alan.le-goff@univ-grenoble-alpes.fr [b] U. Contaldo, C. Cavazza Univ. Grenoble Alpes, CEA, CNRS, IRIG, CBM, F-38000 Grenoble, France E-mail: christine.cavazza@cea.fr

Synthetic procedures for AcPyTACN:

Compound 2 (225m g 0.49 mmol) was dissolved in 5M HCl (5 ml) and the solution refluxed for 3h. Removal of the solvent gave the deprotected AcPyTACN as a green solid.

Preparation of the electrodes

The working electrodes were glassy carbon and gas diffusion electrodes (3 mm diameter). N-methyl-2-pyrrolidinone (NMP) dispersion of MWCNTs were prepared by 30 minutes sonication of 5 mg MWCNTs dispersed in 1 mL NMP until homogeneous black suspension was obtained. Then 20 µL of the MWCNTs solution were drop-casted on a GCE/GDE and NMP was removed under vacuum leaving a 5-µm-thick film on the GCE.

Functionalisation of MWCNT with AcPyTACN or 1-pyrenebutyric acid adamantyl amide and enzyme immobilization

MWCNT-modified electrodes were soaked for 30 minutes in DMF solution containing 10 The differences between the WT enzyme and the C45G/T50C mutant were further investigated at the catalytic level via electrochemical and biochemical approaches. First, the O2 inhibition tests were performed by direct electrochemistry as described for the other CODHs (Chapter 1 section 4.4.3). However, the results revealed no major differences between the two enzymes. Then, the behavior of the WT and the C45G/T50C mutant produced anaerobically and purified anaerobically or aerobically was studied.

Several differences were observed when the WT and the C45G/T50C mutant were purified aerobically, and compared to the WT enzyme purified anaerobically. Firstly, the metal content is 20% and 10% Ni/monomer and 75 % and 60 % of Fe, respectively. Secondly, the WT and C45G/T50C mutant purified aerobically retained only 30 % (~130 U•mg -1 ) and 10 % (~40 U•mg -1 ) of specific CO oxidation activity, respectively.

Furthermore, these two aerobically prepared enzyme samples were stored at 4 °C under aerobic conditions and their activity was controlled over time. After 1 day, The C45G/T50C mutant retains only 5 % (~20 U•mg -1 ) of specific CO oxidation activity, while the WT in the same conditions maintain 25 % of activity (~100 U•mg -1 ) after 8 days.

Taken together, these results strongly suggest a role of the [2Fe2S] D-cluster in DvCODH in O2 protection.

Interestingly, several functions could therefore be attributed to the D-cluster:

■ A role in electron transfer.

■ Due to its position at the dimer interface, the D-cluster could help with dimer stabilization.

■ Depending on its nature, this cluster would provide to the enzyme a better resistance towards O2.

We then decided to start to investigate the role of the D-cluster in RrCODH, with the aim of improving its catalytic performances. During the studies, I had the opportunity to supervise a Master student Emma Colombari (this internship is about 2 months and have to be done at the end of the first year of the master program). A part of her results are presented in this chapter.

These results revealed the appearance of the first brown protein crystals with CaCl2 and PEG 8k concentrations below 0.3 M and 10%, respectively. Based on these results we set the same range of PEG 8k concentration, with lower concentration of CaCl2. The results are reported in Table 9.

Table 9 Crystallization conditions tested for RecRrCODH at 25 mg•mL -1 , mixed 50/50 with the crystallization solution. The results reported in Table 9 show a precipitation gradient of RecRrCODH. Therefore, we decided to test different percentage of MPD (Table 10). Two enzyme concentrations were tested (25 and 17.5 mg•mL -1 ). The first monocrystals of RecRrCODH were obtained with the A1 precipitation conditions of TrisHCl 0.1 M, pH 7.5, DTT 1 mM, CaCl2 0.1 M, PEG 8k 6% and MPD 6%.

-Perspectives

These first results encourage us to pursue the characterization of the RrCODH D-cluster mutants. First of all, in order to better understand the possible role of the D-cluster in [NiFe]-CODHs, we plan an EPR characterization under anaerobic and aerobic conditions of the metalloclusters of the RrCODH D-cluster mutants. Second, we will seek to investigate the impact of the different mutations on the structure/activity relationships through X-ray crystallography. Furthermore, these results raise up the attention of

-Material & Methods

Cloning and expression.

The plasmids harboring the native cooS (WT) (pCDF-Duet1-LIC1_RrCooS) and cooCTJ genes (pRSF_RrCooCTJ) were obtained as previous described (Chapter 2). Regarding the R.r. cooS mutants Dr. Julien Perard cloned the C49S, C49T, C41S, C41A/C49S and C41S/C49S mutants from the native Rr cooS gene and the synthetic G44C/C49T gene was obtained from GenScript. All cooS genes were cloned in the pCDF-Duet1-LIC1 plasmid which contains a TEV cleavage site downstream the histidine tag at the N-terminus (662 residues, 69547 Da for WT).

E. coli BL21 (DE3) or E. coli Rosetta2 ΔiscR (for aG44C/C49T ΔiscR ) were co-transformed with pRSF_RrCooCTJ and pCDF-Duet1-LIC1_RrCooS and cultured under aerobic conditions in auto-induction medium, supplemented with appropriate antibiotics, 1 mM Fe(II)SO4 (Sigma Aldrich), 2 mM L-Cysteine (Sigma Aldrich) and 0.5 mM NiSO4 (ACROS ORGANICS, omitted for aG44C/C49T ΔiscR ), as previous described in chapter 2. E. coli BL21 (DE3) cotransformed with pRSF_RrCooCTJ and pCDF-Duet1-LIC1_RrCooS-G44C/C49T were cultured under anaerobic conditions (anG44C/C49T ΔiscR ) as previously described for the recombinant ChCODH II and IV (J. H. Jeoung & Dobbek, 2007) [START_REF] Domnik | CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2[END_REF]. In this latter case, the expression was induced by addition of 0.2 mM IPTG supplemented with 0.5 mM NiSO4 (ACROS ORGANICS), 1 mM Na2S (Sigma Aldrich), 1 mM Fe(II)SO4 (Sigma Aldrich) once the culture reached an OD600 of 0.5. Bacteria were first cultivated aerobically prior to induce the recombinant expression cultures under anaerobic conditions. Cultures were centrifuged and cell pellets collected under argon flow before being stored in liquid nitrogen.

Protein Purification.

All RrCODH D-cluster mutants and WT enzymes were purified following our previously described protocol, which led to the obtention of pure preparations of almost all enzymes (except aG44C/C49T and anG44C/C49T).

the electrode surface expressed in cm 2 which for a glassy carbon electrode with a diameter of 3 mm correspond to 0.071 cm 2 .

TOF: the Turnover Frequency (also termed kcat) is defined as the maximum number of chemical conversions of substrate molecules per second. It is calculated according to:

𝑇𝑂𝐹 = 𝐼 𝑚𝑎𝑥 𝐹𝑛𝛤 𝑚𝑎𝑥

Where Imax is the maximum current density measured, expressed in A•cm -2 .

E1/2: Half Wave potential of the reversible Cred1/Cred2 redox system of the different Dcluster RrCODH mutants at pH 8.5 was calculated by the average of the peak potentials of the redox couple.

Protein crystallization under anaerobic conditions.

In order to remove the His tags, RecRrCODH were incubated in presence of TEV protease (his-tagged) 1:50 overnight at 10 °C, thus the sample was loaded on NTA-Ni and the RecRrCODH without his tags was collected as not bound. Then the buffer of RecRrCODH was exchanged in 100 mM TrisHCl, pH 7.5 and DTT 1 mM by using a Superdex 200 10/300 Increase size exclusion column. To further confirm the removal of the His tag a dot-blot analysis using anti-His tag antibody coupled to HRP was carried out. No signal was detected for the RecRrCODH without His tag also after long time exposure, with a detection limit in the range of nanomolar. Finally, the enzymatic preparations were concentrated to 25 and 17.5 mg•mL -1 . Crystallization drops were made by diluting 1.5 µL of RecRrCODH with 1.5 µL of a precipitating solution (total reservoir of 0.5 mL).

Crystallization trials were performed under argon in a glove box (Jacomex, O2 < 2 ppm) at 21 °C using the vapor diffusion technique (hanging-drop method), equilibrating the drop against 0.5 mL of the precipitating solution using EasyXtal® 15-Well Tools (Qiagen).

Protein precipitation, nucleation or crystallization occurs from 1 day to 1 month.

I miss one of the most important women in my life, Tina.

"No man succeeds without a good woman behind him. Wife or mother, if it is both, he is twice blessed indeed". [Godfrey Winn]

What you have done for me over the years I still can't get it done. Even if you think you are weak and defenseless, you are the opposite, you had the strength to do it all by yourself, you did not have a French mother like me. You supported me during the first Covid-19 lockdown when perhaps at that moment you were the one who needed it most. You have been constant in supporting my daily life, especially in recent times. I am really happy to share everything with you: thoughts, ambitions, interests such as sport, art and above all music and dance together.

I want to conclude these thanks with the lyrics of a song, since without Music my life would be empty.

[