Philippe Négrel
email: p.negrel@brgm.fr

Romain Millot

Behaviour of Li isotopes during regolith formation on granite (Massif Central, France): Controls on the dissolved load in water, saprolite, soil and sediment

Keywords: weathering, isotopes, lithium, groundwater, soil, sediment, granite

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

mineral water generally has isotopic compositions of 0 to +15‰ (Millot and Négrel, 2007;Millot et al., 2010b).

In the context of water/rock interactions, numerous studies have clearly shown that Li isotopes are a promising proxy for tracing silicate weathering reactions (e.g. Huh et al., 1998[START_REF] Huh | Lithium isotopes as a probe of weathering processes: Orinoco River[END_REF][START_REF] Pistiner | Lithium-isotope fractionation during continental weathering processes[END_REF][START_REF] Kisakurek | Behaviour of Li isotopes during continental weathering: the Bidar laterite profile, India[END_REF][START_REF] Rudnick | Extreme lithium isotopic fractionation during continental weathering revealed in saprolites from South Carolina[END_REF][START_REF] Pogge Von Strandmann | Lithium, magnesium and silicon isotope behavior accompanying weathering in a basaltic soil and pore water profile in Iceland[END_REF]Millot et al., 2007Millot et al., , 2010aMillot et al., , 2010b;;[START_REF] Vigier | The relationship between riverine lithium isotope composition and silicate weathering rates in Iceland[END_REF][START_REF] Wang | Behavior of lithium isotopes in the Changjiang River system: Sources effects and response to weathering and erosion[END_REF][START_REF] Clergue | Influence of atmospheric deposits and secondary minerals on Li isotopes budget in a highly weathered catchment, Guadeloupe (Lesser Antilles)[END_REF]. Silicate minerals host Li and chemical weathering induces isotopic fractionation. This results in enrichment of the heavy isotope ( 7 Li) in solution, the light isotope ( 6 Li) being preferentially retained in secondary weathering mineral phases [START_REF] Huh | Behavior of lithium and its isotopes during weathering of Hawaiian basalt[END_REF][START_REF] Vigier | Quantifying Li isotope fractionation during smectite formation and implications for the Li cycle[END_REF][START_REF] Weynell | Lithium isotopes and implications on chemical weathering in the catchment of Lake Donggi Cona, northeastern Tibetan Plateau[END_REF], similar to other non-traditional stable-isotope systems such as Ca, Fe and B [START_REF] Sullivan | CZ-tope at Susquehanna Shale Hills CZO: Synthesizing multiple isotope proxies to elucidate Critical Zone processes across timescales in a temperate forested landscape[END_REF]. Today, lithium isotopes are applied to a wide range of geochemical processes, such as weathering [START_REF] Pistiner | Lithium-isotope fractionation during continental weathering processes[END_REF][START_REF] Huh | Behavior of lithium and its isotopes during weathering of Hawaiian basalt[END_REF][START_REF] Hathorne | Temporal record of lithium in seawater: A tracer for silicate weathering?[END_REF][START_REF] Lemarchand | Lithium isotope systematics in a forested granitic catchment (Strengbach, Vosges Mountains, France)[END_REF][START_REF] Wanner | On the use of Li isotopes as a proxy for water-rock interaction in fractured crystalline rocks: A case study from the Gotthard rail base tunnel[END_REF][START_REF] Wimpenny | The behaviour of Li and Mg isotopes during primary phase dissolution and secondary mineral formation in basalt[END_REF]Ryu et al., 2015;[START_REF] Pogge Von Strandmann | Experimental determination of Li isotope behaviour during basalt weathering[END_REF]. Other applications are to the study of erosion processes, either in large river-basin studies (Huh et al., 1998[START_REF] Huh | Lithium isotopes as a probe of weathering processes: Orinoco River[END_REF][START_REF] Kisakurek | Li and δ 7 Li in Himalayan rivers: Proxies for silicate weathering? Earth Planet[END_REF]Millot et al., 2010a;[START_REF] Dellinger | Riverine Li isotope fractionation in the Amazon River basin controlled by the weathering regimes[END_REF][START_REF] Henchiri | The influence of hydrothermal activity on the Li isotopic signature of rivers draining volcanic areas[END_REF]2016;[START_REF] Wang | Behavior of lithium isotopes in the Changjiang River system: Sources effects and response to weathering and erosion[END_REF][START_REF] Murphy | Tracing silicate weathering processes in the permafrostdominated Lena River watershed using lithium isotopes[END_REF], or in smaller streams [START_REF] Vigier | The relationship between riverine lithium isotope composition and silicate weathering rates in Iceland[END_REF][START_REF] Pogge Von Strandmann | Assessing the role of climate on uranium and lithium isotope behaviour in rivers draining a basaltic terrain[END_REF][START_REF] Liu | Processes controlling δ 7 Li in rivers illuminated by study of streams and groundwaters draining basalts[END_REF]; to studying anthopogenic disturbance in soils (Négrel et al., 2010;[START_REF] Tsai | Lithium distribution and isotopic fractionation during chemical weathering and soil formation in a loess profile[END_REF]; and as geothermometer proxies in mineral waters (Millot and Négrel, 2007), or for tracing groundwater flowpaths [START_REF] Négrel | Isotopic evidence of lead sources in Loire River sediment[END_REF][START_REF] Pogge Von Strandmann | Chemical weathering processes in the Great Artesian Basin: Evidence from lithium and silicon isotopes[END_REF].

However, studies on granitic bedrock and its associated weathering products are very scarce [START_REF] Rudnick | Extreme lithium isotopic fractionation during continental weathering revealed in saprolites from South Carolina[END_REF][START_REF] Lemarchand | Lithium isotope systematics in a forested granitic catchment (Strengbach, Vosges Mountains, France)[END_REF].

At the basic scale of a watershed, the challenge is to integrate all the reservoirs and parts of the watershed affected by the weathering processes. This has been the case in studies involving classic tools, such as strontium isotopes, but not when working with newly developed tools. In this study, we measured lithium and its isotope values in all reservoirs and parts of a catchment draining granite. This included surface-and mineral waters, primary minerals and accessory phases of the granites, the whole rock, and samples of soil and sediment generated within the watershed, completed with additional data for rainwater.

Our study has used Li-isotope data for tracing silicate weathering processes at the basic scale of a watershed, considering all its components. This is a novel approach for Li isotopes, which earlier were partly limited due to the difficulty of collecting and analysing samples from all reservoirs. This systematic work is part of a long-term, wide-ranging and integrated geochemical study covering major and trace elements, Sr isotopes, Nd isotopes and rare earth elements, in water, sediment and soil within the Margeride mountains of central France [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF][START_REF] Négrel | Rare Earth Elements, neodymium and strontium isotopic systematics in mineral waters: evidence from the Massif Central, France[END_REF]; this also includes incorporation of all relevant outside data, such as on separated minerals and long-term monitoring of rainfall.

Isotopic investigation of weathering products generally provides good information on the chemical reactions taking place during such processes. This is further demonstrated by recent studies on the Li isotope systematics of silicate-rock weathering, in complement to investigations covering regolith production and the development of weathering profiles [START_REF] Vázquez | Regolith production and chemical weathering of granitic rocks in central Chile[END_REF][START_REF] Ackerer | Regolith evolution on the millennial timescale from combined U-Th-Ra isotopes and in situ cosmogenic 10 Be analysis in a weathering profile (Strengbach catchment, France)[END_REF].

In order to apply Li isotopes as effective tracers of water/rock interaction processes in a small mono-lithological catchment, the effect of atmospheric input was investigated to correct the lithium values of surface-and mineral waters. This allowed replacing Li isotopes with Sr isotopes in a larger weathering framework. We also tested a steady-state weathering model and fractionation process modelling on the Margeride granite-gneiss, prerequisites for our reactivetransport model computer simulations that coupled a large number of chemical reactions with mass transport, a key challenge for the effective use of Li isotopes as a tool in weathering studies.

-General setting

The granite-gneiss Margeride massif is located in the south of the French Massif Central (Figs. 1a,b). It consists of light and dark granite-gneiss with an average mineral composition of the light facies of 37% quartz, 30% oligoclase, 23% K-feldspar and 10% biotite, and of 31% quartz, 30% andesine, 20% K-feldspar and 19% biotite in the dark facies (Marchand et al., 1985). The mineralogy and chemistry of Margeride granite and gneiss are similar, although the relative abundance of minerals may fluctuate slightly. The presence of a micaschist unit in the massif is worth noting, composed of quartz, muscovite and biotite. Couturié et al. (1979) reported that the different facies of the Margeride granite (323±12 Ma., Rb-Sr isochron) resulted from fractional crystallization of a crustal magma in a subhorizontal laccolith. The Desges River, a tributary of the Allier, flows approximately 40 km between the upper part of the Margeride massif and its confluence with the Allier [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF]Fig. 1b). The watershed covers 89 km 2 and ranges in altitude from 1250 m (headwaters) to 650 m (outlet); the dense N-S-oriented drainage pattern mainly on the top of the Margeride plateaus receives an average annual precipitation of 1000 to 1200 mm/yr -1 . Figure 1. General location map of the Massif Central (in blue-grey), and detailed map of the Desges watershed showing the sampling sites, modified from [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF]. The main geological units are granite, gneiss, and micaschist. The different sampling points are: bedrock and granitic grit of the Desges watershed, mineral springs (Mazel and Fontaine Basses (M.F) and Ranc (R), surface waters, soils and sediments (Dx).

-Material and methods

In a temperate climate, granite usually weathers into grit [START_REF] Sequeira Braga | Climatic zonality of coarse granitic saprolites ("arènes") in Atlantic Europe from Scandinavia to Portugal[END_REF]2002;[START_REF] Pera | Weathering of gneiss in Calabria, southern Italy[END_REF]Paquet and Clauer, 2012). Unweathered bedrock and granitic grit were collected at the same place (Fig. 1, blue square). The Mazel grit sample, collected on top of a granite outcrop, has a fabric skeleton structure retaining the original granitic one, which is commonly observed in the lower (45-100 cm depth) part of the soil profile here. Although the texture of the parent rock was largely preserved, the main transformations are chloritization of biotite, kaolinization of plagioclase, and the presence of oxy-hydroxides [START_REF] Sarazin | Géochimie de l'aluminium au cours de l'altération des granites et basaltes sous climat tempéré[END_REF]. This is reflected in our sample that is mainly composed of quartz (20%), K-feldspar (20%), plagioclase (20%) and chlorite (5%), and for which we adopt the classification of [START_REF] Begonha | Weathering of the Oporto granite: geotechnical and physical properties[END_REF] as "weathered rock".

Samples A0, A1 and A2 were collected around station D17, and samples B0, B1 and B2 around station D19 (Fig. 1). A0 and B0 [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], collected from a weathering profile, are not a direct skeleton-fabric material of the granite, but are surface saprolite mainly composed of sand and silt. Samples A1 and B1 are riverbank sediments, classified as sandy-silt. All four samples mainly consist of quartz, K-feldspar, plagioclase and illite-micas, with dramatic changes in relative abundances [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF].

The soils in this catchment are podzol and peat, varying in thickness from a few centimetres at the top of the profile to a few metres farther down. They mainly consist of kaolinite, hydroxides and vermiculite, K feldspar and biotite (isolated from the matrix), and ubiquitous organic and amorphous phases (Bourrié, 1978;[START_REF] Sarazin | Géochimie de l'aluminium au cours de l'altération des granites et basaltes sous climat tempéré[END_REF]. Soil samples A2 and B2, collected in fields bordering the streams, can be classified as silty-sand and contain small amounts of quartz, K-feldspar, plagioclase and illite-micas.

The sample-preparation procedure was described in detail by Negrel and Deschamps (1996) and [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF]. Briefly, weathered rock (Mazel) and surface saprolite (A0 and B0) were powdered, the other soil and sediment samples being oven-dried at 70 °C, homogenized, quartered and dry-sieved through a 165 µm nylon mesh, and powdered before analysis. The δ 7 Li values were measured on the sediment-and soil samples following standard acid-dissolution. For rock and sediment, total sample digestion was necessary before separation of Li from the matrix. About 50 mg of crushed sample was dissolved in a closed beaker with an ultrapure mixture of three acids [4 ml HF (23N), 1 ml HNO3 (14N) and 0.1 ml HClO4 (12N)]

for four days at 100 °C. After this, once the acid mixture had evaporated, 4 ml of HCl (6N) was added for four days at 100 °C. A sample aliquot (30 ng of Li) of the acid-dissolution residue was then dissolved in 0.5 ml of HCl (0.2N) before being placed in a column containing cationic resin for Li separation.

Surface-water samples were collected from the river according to the protocol in [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF]; groundwater samples were thermo-mineral waters from naturally flowing springs, taken according to the protocol in [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF]. Briefly, water samples were collected in the field in acid-washed polyethylene bottles after filtration through 0.2 µm Sartorius® cellulose acetate filters and then acidified to pH <2, by adding ultra-pure HNO3.

Lithium isotopic compositions in soil, sediment and water were measured with a Neptune MC-ICP-MS (see Millot et al., 2004, for details) and Li concentrations with a quadrupole ICP-MS. The 7 Li/ 6 Li values were normalized to the L-SVEC standard solution (NIST SRM 8545) following the standard-sample bracketing method. Typical in-run precision on δ 7 Li determination was about 0.1-0.2‰ (2σm). Chemical separation of lithium from the matrix was achieved before mass analysis following a procedure adapted by [START_REF] Millot | Accurate and high precision measurement of lithium isotopes in two reference materials by MC-ICP-MS[END_REF], using cationic resin (Bio Rad AG® 50W-X12, 200-400 mesh) and HCl (0.2N) for 30 ng of lithium. Blanks for total chemical extraction were less than 20-30 pg of Li, which is negligible since the blank/sample ratio is less than 10 -3 . Accuracy and reproducibility of the total method (purification procedure plus mass analysis) were tested by repeated measurements of a seawater standard (IRMM BCR-403) after separation of Li from the matrix, for which we obtained a mean value of δ 7 Li = +30.8‰±0.4 (2σ, n=15) over the duration of the analytical period. This mean value agrees with our long-term measurements (δ 7 Li = +31.0‰±0.5, 2σ, n=30, [START_REF] Millot | Accurate and high precision measurement of lithium isotopes in two reference materials by MC-ICP-MS[END_REF]) and with other values from the literature.

-Results

The Li data (δ 7 Li and Li contents) of rock, grit, soil, sediment and separate minerals from the granite and surface and groundwater collected in the catchment are given in Table 1. Millot and Négrel, 2007; results of Li concentration (µmol l -1 ) and δ 7 Li for surface waters.

For the separate minerals, Li-rich phases decreased in content from biotite (489 μg g -1 )

to K-feldspar (5 μg g -1 ), for an average whole-rock Li content of about 143 μg g -1 . Lithium isotopic compositions in the separate minerals ranged from -2.6‰ in K-feldspar to 0.0‰ in apatite, for a δ 7 Li of -1.9‰ in bulk rock. Reconstituting the whole rock from the separate minerals led to a Li content of 56 μg g -1 and a δ 7 Li of 0.45‰. These values are inconsistent with the measured ones, implying that the rest of the Li probably occurs in residual minerals with a Li content of around 86.6 μg g -1 and a δ 7 Li of -3.43‰. The Li content in soil and sediment fluctuated from 56 μg g -1 (surface saprolite A0) to 111 μg g -1 (soil A2). With the exception of soil A2 (almost twice the content in soil B2), the samples had similar Li contents (surface saprolite 56 vs. 61 μg g -1 ; sediment samples 69 vs. 62 μg g -1 ). Li isotopes in soil and sediment samples ranged from -3.38‰ (soil A2) to 3.31‰ (surface saprolite B0).

Surface waters in the catchment had a roughly constant Li content of around 0.14 to 0.24 μmol L -1 , corresponding to the lower value observed in streams draining volcanic rocks in the Massif Central, (Table 1), whose range of variation is from 0.4 to 2.35 μmol L -1 (Négrel et al., 2010a). Li-isotope compositions (δ 7 Li) varied in the catchment ( al., 2010) that range from +12 to +15.1‰. Lithium-isotope compositions and Li concentrations in the surface waters have a linear relationship (δ 7 Li = 5.36 x Li + 3.73), with a correlation coefficient r 2 = 0.81 (n=6). For groundwater, the lithium concentrations and δ 7 Li were published by Millot et al. (2007): Li concentrations ranged from 157 μmol L -1 (Ranc 1) to 547 μmol L -1 (Mazel; their Table 3), which is one order of magnitude higher than in surface water and consistent with their data for thermo-mineral waters in the Massif Central. Lithiumisotope compositions (δ 7 Li) in these groundwaters were less variable (+2.6 to +5.0‰) than in surface waters, with the lowest value observed in Ranc 2 (+2.6‰) and the highest in Mazel (+5‰), a range of variation similar to that in thermo-mineral waters of the Massif Central (δ 7 Li from +2.6 to +5.5‰, Millot et al., 2007).

-Discussion

Mobility of Li during weathering

In order to evaluate the extent of the chemical mobility of Li during granite weathering, we calculated the weathering intensity with τ, the elemental mass transfer coefficient of [START_REF] Brimhall | Constitutive mass balance relations between chemical composition, volume, density, porosity, and strain in metasomatic hydrochemical systems: results on weathering and pedogenesis[END_REF]. This calculation avoids the dilution effect and impact of volume change due to weathering [START_REF] Nesbitt | Mobility and fractionation of rare earth elements during weathering of a granodiorite[END_REF]. We thus calculated the percentage change for Li relative to the immobile element Ti [START_REF] Clergue | Influence of atmospheric deposits and secondary minerals on Li isotopes budget in a highly weathered catchment, Guadeloupe (Lesser Antilles)[END_REF] with equation ( 1), previously used for other elements in the same catchment [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF] and according to [START_REF] Nesbitt | Mobility and fractionation of rare earth elements during weathering of a granodiorite[END_REF]:

τLi= 100 × ([(Li/Ti)sample/(Li/Ti)granite] -1)
(Equation 1)

Expressed as a percentage, τ = 0 represents the initial unweathered material and τ = -100% means complete removal of Li from the parent material. A positive value reflects Li enrichment by concentration in residual minerals and negative values represent Li depletion, lost from bedrock because of its solubility. However, compared to other soluble elements, less depletion of Li may be related to its incorporation into secondary minerals. The percentage change of Li in the samples, i.e. loss or gain of mobile Li relative to immobile Ti (τLi, Fig. 2), ranges from -31 to -66% , the extreme values being for surface-saprolite samples (A0, B0). The grit is depleted around -47% compared to bedrock, with a decrease of the δ 7 Li value and a Li content from -1.9‰ and 143.1 µg g -1 in granite to -3.4‰ and 104.7 µg g -1 in grit (Fig. 3). The D19 samples show more Li depletion in surface saprolite (A0, -66%) than in riverbank sediment (A1, -60%) and soil (A2, -46%). This depletion is marked by a continuous decrease of δ 7 Li values from +0.1‰ in surface saprolite to -3.4‰ in soil. Compared to the decrease in δ 7 Li values, Li concentration remains constant between samples A0 and A1, and shows an increasing trend in soil sample A2 (Fig. 3), which has the same δ 7 Li and Li-concentration values as those of the grit. 1).

The mineralogical composition of the samples was earlier presented in [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF].

Briefly, surface saprolite A0 is composed of quartz (35%), K-feldspar (~20%), plagioclase (~20%) and illite-micas (~20%). At station D19, riverbank-sediment sample A1 is quite different, with more quartz (50%), less feldspars (5%) and illite-mica around 20%; it is also richer in chlorite (20%). Soil sample A2 contains the least quartz (15-20%); with K-feldspar, illite-micas and plagioclase all around 20%. This variation is accompanied by more negative δ 7 Li values, meaning that Li is enriched in soil with a fractionation of Li isotopes and changes in the mineral abundance of the samples (residual minerals have negative δ 7 Li; Fig. 3). This is accompanied by a change in the Li concentration, with an 18% increase between A0 and A1, from 56 to 69 μg g -1 , and a38% increase between A1 and A2, from 69 to 112 μg g -1 . Between saprolite and soil, the Li concentration increases by 50%. Surface saprolite B0, depleted around -31%, has a positive δ 7 Li value of +3.3‰, clearly higher than all mineral values in the granite. Sediment sample B1 is largely depleted (-60%) with a δ 7 Li value close to 0‰. Soil sample B2 is depleted around -50% with a more negative δ 7 Li value of -2.7‰. Compared to the significant variation in δ 7 Li values, the Li concentration remains largely constant in the three samples with a total variation of around 8% (Fig. 3). Quartz reaches 65% in surface saprolite sample B0, K-feldspar and illite-smectite having a similar abundance (~20%), but plagioclase is only 5%. Riverbank sediment sample B1 contains around 25% quartz, and 20% K-feldspar, 20% plagioclase and 20% illite-mica, but only 5% chlorite with 15% as an amorphous phase. Soil sample B2 contains the least quartz (15-20%), but Kfeldspar, illite-micas and plagioclase contents are still around 20%. Again, the δ 7 Li values become more negative from surface saprolite to soil, which can be explained by changes in mineral abundance in the samples, as higher amounts of residual minerals in the soil sample lead to more negative δ 7 Li values.

Atmospheric input correction to surface and groundwater

The need to quantify and subtract the portion of elements contributed by precipitation in the chemical composition of surface-and ground-waters has been apparent for several decades (e.g., [START_REF] Stallard | Geochemistry of the Amazon: 1. Precipitation chemistry and the marine contribution to the dissolved load at the time of peak discharge[END_REF][START_REF] Meybeck | Atmospheric inputs and river transport of dissolved substances[END_REF]. Basically, quantifying the precipitation input requires knowledge of its chemical composition [START_REF] Négrel | Erosion sources determined from inversion of major, trace element ratios and strontium isotopic ratios in river water: the Congo Basin case[END_REF]Grobois et al., 2000).

The classic reference of atmospheric input in unpolluted hydrosystems is chloride, which behaves conservatively throughout the hydrological cycle; the correction for its atmospheric contribution is estimated by reference to the Cl concentration in river water, multiplied by the ratio element/Cl of precipitation (e.g., [START_REF] Meybeck | Atmospheric inputs and river transport of dissolved substances[END_REF][START_REF] Négrel | Erosion sources determined from inversion of major, trace element ratios and strontium isotopic ratios in river water: the Congo Basin case[END_REF]. As stated by [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], in the absence of evaporites in the Margeride catchment, Cl ions in the river water can be assumed to originate from wind-blown sea salt and human activity. High Cl contents can be found in (thermo-mineral) groundwater emerging as naturally flowing springs, which are closely related to fractures in the granite that cannot play any role in supplying Cl to the river water. This would have been different in the case of an unexposed basement rock with a cover of sedimentary rock, as described by Négrel et al. (1997). In our mass-balance approach, determining input-output budgets for dissolved constituents in waters similar to [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF] work, we used the mean weighted rainfall on the Massif Central over 1.5 years for Cl content [START_REF] Négrel | Rain chemistry in the Massif Central (France). A strontium isotopic and major elements study[END_REF].

To determine the highest chloride concentration derived from precipitation input, the standard method [START_REF] Meybeck | Atmospheric inputs and river transport of dissolved substances[END_REF][START_REF] Négrel | Erosion sources determined from inversion of major, trace element ratios and strontium isotopic ratios in river water: the Congo Basin case[END_REF] is to multiply the mean weighted Cl value by a concentration factor F resulting from evapotranspiration. With a mean weighted chloride of around 20 µmol L -1 and an F value of approximately 1.6, around 32 µmol L -1 represent the highest chloride concentration originating from atmospheric input to the river.

When the Cl content in water is below 32 µmol L -1 (Table 1), the entire chloride content of the river is assigned an atmospheric origin; when it is over 32 µmol L -1 , the residual chloride in the river is attributed to human activity [START_REF] Meybeck | Atmospheric inputs and river transport of dissolved substances[END_REF].

Using rainwater samples obtained during a rain-input survey over the Massif Central [START_REF] Négrel | Rain chemistry in the Massif Central (France). A strontium isotopic and major elements study[END_REF], Millot et al. (2010b) reported low lithium concentrations, between 0.004 and 0.292 µmol L -1 , whereas the δ 7 Li values varied greatly between +3.2‰ and +95.6‰

over the study period. From this observed range, two groups of rainwater with very different Li characteristics were identified (Fig. 4). The first one, named "natural", had a Li concentration of 0.04 µmol L -1 with a δ 7 Li value of around +6.9‰; the second one, named "anthropogenic", had a Li concentration of 0.13 µmol L -1 and a much higher δ 7 Li value of around +79‰. For the Li isotopes, considering natural and anthropogenic components, the average value over the 1.5 year survey was +26.2‰ and the mean concentration-weighted Li isotopes value was +20.3‰ (Millot et al., 2010b). For correcting Li contents and Li isotopes of surface water from rainfall data, we used the average Li content, 0.06 µmole L -1 , and the average δ 7 Li, +26.20‰, of all rainwater samples collected in the Massif Central over 1.5 years [START_REF] Négrel | Rain chemistry in the Massif Central (France). A strontium isotopic and major elements study[END_REF]Millot et al., 2010b;Fig. 4). (1999; 2006) for anion contents and now, in this study, for Li (Fig. 4). Applying the atmospheric correction to these surface-water samples showed that D9b and D8 derived their total Cl content from precipitation input and thus reflect no anthropogenic influence, with 35% and 61% of the Li deriving from atmospheric input, respectively. This shows a non-negligible influence of precipitation input on the Li budget in this case, contrary to other observations at very different scales by [START_REF] Ryu | Variation of lithium isotope geochemistry during basalt weathering and secondary mineral transformations in Hawaii[END_REF] in Hawaii, or by Millot et al. (2010) in the Mackenzie Basin of northern Canada.

Other samples have a residual Cl content that is generally related to anthropogenic input, mainly from agricultural activity. Here, the Li derived from atmospheric input ranged from 40% in sample D11 to 43% in samples D9 and D10. Accordingly, as done by [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF] for the same catchment, we corrected the Li-isotope values observed in the water for atmospheric contribution, which gave a δ 7 Li ranging from +10.8 (D11') to -2.0‰ (D8). The δ 7 Li for samples without residual Cl are the lowest, with a δ 7 Li of -2.0‰ for sample D8 and +0.8‰ for sample D9b. A similar atmospheric correction to mineralized waters has a low impact on Li content (less than 0.06% of the Li being from precipitation input) with no change in the δ 7 Li value, indicating the dominant influence of water/rock interaction on both Li content and δ 7 Li value of the mineralized waters.

However, contrary to previous work on atmospheric and anthropogenic corrections to surface-water chemistry [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF][START_REF] Petelet-Giraud | Variability of 87 Sr/ 86 Sr in water draining granite revealed after a double correction for atmospheric and anthropogenic inputs[END_REF], we cannot use the same method for anthropogenic-input correction here. The residual Cl after atmospheric correction derives from anthropogenic input and the correction for such input (e.g. to a zero Cl content) requires knowledge of its Li/Cl ratio. However, this ratio cannot be constrained accurately, which would allow a robust correction for Li content and thus for Li isotopes.

Applying the atmospheric correction to the D8, D9, D10 and D11 surface water samples [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], shows that D8 and D9 derived their total Cl content from atmospheric input, and thus reflect no anthropogenic influence. The Sr content derived from atmospheric input is around 50% in D9 and 57% in D8. The influence of atmospheric input on 87 Sr/ 86 Sr ratios is consistently significant [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], and, corrected for atmospheric input, the 87 Sr/ 86 Sr ratios for water-granite interaction range from 0.72316 (D8) to 0.72511 (D9). Once corrected for atmospheric input, the δ 7 Li value and Cl concentration are controlled by water-granite interaction and anthropogenic input(s). This is summarized in Fig. 5, where δ 7 Li is plotted versus residual Cl. The two samples with no residual Cl directly yield the value of the δ 7 Li derived from water-rock interaction, with δ 7 Li values of +0.8‰ (D9) and -2.0‰ (D8) associated with Li concentrations of 0.11 and 0.06 µmol L -1 , respectively. The other samples have residual Cl values from 7.4 (D10) to 49.7 µmol L -1 (D11), δ 7 Li value from +6.4‰ (D9) to +10.8‰ (D11) and nearly constant Li concentrations of 0.13 to 0.15 µmol L -1 . This can be related to an increase of the anthropogenic input, with a roughly constant δ 7 Li.

Regarding the anthropogenic Li sources, Négrel, et al. (2010) gave a range for the δ 7 Li in fertilizers, such as carbonaceous amendment or NPK, between +2.1‰ and +215‰ and a mean weighted δ 7 Li value of +14.2‰. For Li used with other trace elements, such as Cu, Fe, Mn, Zn, Co, Mo and B, even though not necessary as an additional nutrient (Négrel et al., 2010a), the δ 7 Li ranged between -16‰ and + 31‰ with a mean weighted δ 7 Li value of +1.3‰.

These two mean values agree with the range observed in surface waters and thus can explain an anthropogenic input from agricultural activities. [START_REF] Négrel | Strontium isotope systematics used to decipher the origin of groundwaters sampled from granitoids: the Vienne case (France)[END_REF] developed a weathering model based on dissolution, giving the theoretical (th) isotopic signature (i) of a chemical element (Z) in water being in equilibrium with granite (iZth), which was initially developed for and applied to strontium. This model considers the congruent dissolution of Sr-bearing phases (mx), their abundance (%mx), isotope ratio (iZmx) and element concentration (Zmx). Each mineral characteristic considered (iZmx and Zmx) is weighted by the weatherability (Wmx) of each [START_REF] Négrel | Strontium isotope systematics used to decipher the origin of groundwaters sampled from granitoids: the Vienne case (France)[END_REF], summarized in equation 2: 2)

The weathering model for Li

The simple dissolution model

( ) ( ) ∑ ∑ × × × × × = mx mx mx mx mx mx mx th W Z W Z iZ iZ % % (Equation
The theoretical isotopic composition (iZth) of water in equilibrium with granite was then used for Sr in several environments [START_REF] Négrel | Strontium isotope systematics used to decipher the origin of groundwaters sampled from granitoids: the Vienne case (France)[END_REF][START_REF] Petelet-Giraud | Variability of 87 Sr/ 86 Sr in water draining granite revealed after a double correction for atmospheric and anthropogenic inputs[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], before being redefined and applied to Nd and Pb in the present watershed [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF]Négrel et al., 2010b). The initial model was based on the hypothesis that the dissolution of plagioclase, K-feldspar and biotite contributes most elemental Sr. The same set of minerals, plus apatite for Nd, and apatite and allanine for Pb, were then considered [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF]Négrel et al., 2010b). For lead-isotope compositions during granite dissolution, we used the concept of [START_REF] Harlavan | The release of Pb and REE from granitoids by the dissolution of accessory phases[END_REF] that highlights the role of accessory phases and primary minerals in controlling the isotope composition during water-rock interaction; for the accessory phases, it was suggested that apatite played a major role. Our model thus assumes that only the fluid concentration changes, the isotopic ratio remaining constant, because the secondary minerals are in equilibrium with the parent solution for Sr, Nd and Pb. The relative mineral weatherability Wmx, considering that of plagioclase as 1, is 0.25 for biotite and 0.1 for Kfeldspar [START_REF] Négrel | Strontium isotope systematics used to decipher the origin of groundwaters sampled from granitoids: the Vienne case (France)[END_REF]. A sensitivity analysis of the model (Petelet-Giraud et al., 2003b;[START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], based on testing various mineral-weatherability values from the literature (Blum et al., 1994;Zuddas et al., 1995), consistently showed only minor differences in the isotope values. The model was verified for Sr by comparing the iSrth calculation for given conditions of Sr abundance and the isotopic characteristics of separate minerals, with the 87 Sr/ 86 Sr ratio of water that has interacted with several granitoids and basalts in France [START_REF] Petelet-Giraud | Variability of 87 Sr/ 86 Sr in water draining granite revealed after a double correction for atmospheric and anthropogenic inputs[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF].

For this study, the model was adapted for lithium and applied to the Margeride granite, using data from Table 1. In view of the composition of this granite, we considered K-feldspar, plagioclase and biotite as primary minerals, and apatite as an accessory phase in the model of eq. 2. This was also based on interaction experiments by [START_REF] Millot | Chemical weathering of granitic rocks: experimental approach and Pb-Li isotopes tracing[END_REF] between a rainwater reference solution (TMRAIN-95) and 1.5 g of powdered granite sample from India at 25 °C (Négrel et al., 2010b). [START_REF] Millot | Chemical weathering of granitic rocks: experimental approach and Pb-Li isotopes tracing[END_REF] monitored Pb-and Li-isotopic compositions over 12 months of interaction, showing that: i) When using Pb isotopes, there is a major contribution of K-feldspar and plagioclase dissolution, and, to a lesser extent, of biotite and apatite dissolution;

ii) When using Li isotopes, the light lithium isotope ( 6 Li) is preferentially retained during uptake of Li into secondary minerals during weathering.

The theoretical δ 7 Li signature for water draining this granite is close to -3.3‰. The difference in the isotopic values is always less than 0.1 for Li during sensitivity analysis of the model, done by testing various mineral-weatherability values similar to the Sr, Nd and Pb models [START_REF] Petelet-Giraud | Variability of 87 Sr/ 86 Sr in water draining granite revealed after a double correction for atmospheric and anthropogenic inputs[END_REF][START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF]Négrel et al., 2010b). Our model only provides information on the isotopic value of water after interaction with minerals; it does not assume that the main process associated with isotope fractionation is the formation of secondary precipitates, such as clays, even though the evolution of Li-isotope values is controlled by the precipitation of secondary minerals, as was shown in other environments [START_REF] Huh | Lithium isotopes as a probe of weathering processes: Orinoco River[END_REF][START_REF] Pistiner | Lithium-isotope fractionation during continental weathering processes[END_REF][START_REF] Vigier | Quantifying Li isotope fractionation during smectite formation and implications for the Li cycle[END_REF]Millot et al., 2010).

A broader weathering frame using Li and Sr isotopes: Implications for water/rock interaction in the critical zone

Figure 6 is a plot of the δ 7 Li vs. 87 Sr/ 86 Sr values, where the results from primary minerals and accessory phases of the granites, the whole rock, the soil and sediment samples, and the mineralwater and surface-water samples, are plotted with the results of the weathering-model computation for the granite. The Sr-isotope systematics for primary minerals and accessory phases of the granites, whole rock, soil and sediment samples, and mineral-water data are from [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], and those for surface-water samples are from [START_REF] Négrel | Geochemical study of a granitic area -The Margeride Mountains, France: chemical element behavior and 87 Sr/ 86 Sr constraints[END_REF].

Figure 6 shows several features: 1) For surface waters, the δ 7 Li and 87 Sr/ 86 Sr values disagree with the weathering model, even if Li is not corrected for atmospheric input, the δ 7 Li being either lower or higher than the model value. 2) For thermo-mineral spring waters, there is again no agreement with the results of the weathering model, all springs having a higher δ 7 Li than the weathering model, and a larger influence caused by an increase in weathering of one or more minerals cannot explain the values observed for δ 7 Li values in the mineralized waters, no minerals in the Margeride granite having such a high δ 7 Li value. This is particularly true for the residual trace minerals that cannot control the δ 7 Li values of the water (Figs. 3 and6). For thermo-mineral spring waters, the δ 7 Li values thus suggest that other processes lead to the observed signatures because the 87 Sr/ 86 Sr ratio agrees with the results of the weathering model.

3) For the residual solids, the 87 Sr/ 86 Sr difference between whole rock (0.72269) and Mazel grit (0.73134) reflects the weathering of low 87 Sr/ 86 Sr-and Rb/Sr-bearing minerals, such as plagioclase and K-feldspar; this is confirmed by the grit mineralogy (~20% of each in this sample; [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], whereas the Mazel grit shows a depleted δ 7 Li compared to the whole rock. 4) Riverbank sediment A1 at site D19 shows an 87 Sr/ 86 Sr ratio of 0.74408 and a δ 7 Li of 0.1‰, both higher than that of the grit. An increase in the Sr-isotope ratio is noted in the accompanied by a large decrease in δ 7 Li (from +0.1 to -3.4‰). In site D17, soil B2 has the lowest 87 Sr/ 86 Sr ratio (0.76296) and δ 7 Li (-2.7‰), and, as for site D19, the 87 Sr/ 86 Sr (0.76649) in sediment B1 collected on the river bank increases to 0.78508 in surface saprolite B0, accompanied by a large increase in the δ 7 Li (from 0 to +3.3‰).

Modelling Li isotopic fractionation using a Raleigh equation: the role of secondary phase precipitation

Here, we apply the batch fractionation model for Li isotopes as developed by [START_REF] Bouchez | Modeling novel stable isotope values in the weathering zone[END_REF] and used by [START_REF] Clergue | Influence of atmospheric deposits and secondary minerals on Li isotopes budget in a highly weathered catchment, Guadeloupe (Lesser Antilles)[END_REF]. This model is based on the non-congruent dissolution of bedrock leading to a δ 7 Li in solution (δ 7 Lisolution). As a first approach, this model is only based on (non congruent) dissolution/precipitation reactions. The main process occurring in this solution is Li-isotope fractionation through the formation of secondary phases that preferentially incorporate light lithium ( 6 Li). This gives a residual solution with a δ 7 Lidiss and a δ 7 Lisec for the secondary phases. The δ 7 Lisec can be expressed as follows:

(Equation 3)

The key parameters are the isotope fractionation factor δ 7 Lisec between the different secondary minerals, and the solution (δ 7 Lisolution) and proportion (1-fLi) of Li in secondary phases. The fLi can be expressed by the elemental mass transfer coefficient τ calculated with equation ( 1), which represents the evolution of the Li/Ti ratio in sediment compared to that of bedrock.

The isotope fractionation factor is calculated from chemical and isotope data measured on field material; this can be applied to samples from soil, rivers and the sedimentary record, and does not require prior knowledge of the isotope fractionation factors involved in the detailed reactions. Weathering products are modelled here with a fractionation factor αsec-dis ranging from 0.975 to 0.998, consistent with the range of α values used in the literature [START_REF] Rudnick | Extreme lithium isotopic fractionation during continental weathering revealed in saprolites from South Carolina[END_REF][START_REF] Clergue | Influence of atmospheric deposits and secondary minerals on Li isotopes budget in a highly weathered catchment, Guadeloupe (Lesser Antilles)[END_REF]. To summarize, the α values in the literature span a range from 0.970 to 1.000 and derive either from laboratory experiments (0.979-0.984, Millot et al., 2010c;[START_REF] Vigier | Quantifying Li isotope fractionation during smectite formation and implications for the Li cycle[END_REF], or from field data (0.982-0.989, Millot et al., 2010aMillot et al., , 2010c;;Tipper et al., 2012;[START_REF] Dellinger | Lithium isotopic composition of the dissolved load in the Amazon River Basin[END_REF]2014;2015).

We found that the fractionation factor αsec-dis on Figure 7 can explain some, but not all, weathering products; the lowest δ 7 Li observed in the samples fit with the α max (0.998), but no point is explained by the α min (0.975). Thus, we cannot fit the required fractionation factor αsec-dis to the soil and sediment data (Fig. 7). Additional processes or inputs should thus be considered and investigated.

Facing a broader range of isotopic fractionation for the precipitation of secondary phases than the one defined from experimental clay precipitation, [START_REF] Lemarchand | Lithium isotope systematics in a forested granitic catchment (Strengbach, Vosges Mountains, France)[END_REF] argued for the precipitation of another possible phase with a higher isotope fractionation. As far as we know, no studies have been carried out on such phases, but oxides could be a good candidate for the fractionation of Li isotopes and can account for the existence of high δ 7 Li values in weathering products. This argument was already proposed by Millot et al. (2010a) to explain incipient weathering effects on Li isotopes in the Canadian Rocky Mountains. Concerning our samples, only B1 and B2 have an amorphous phase identified by X-ray diffraction (15 and 5%, respectively; [START_REF] Négrel | Water-granite interaction: clues from strontium, neodymium and rare earth elements in saprolite, sediments, soils, surface and mineralized waters[END_REF], agreeing with a δ 7 Li shift away from the α max line, but only for B1, as sample B2 plots close to the α max line. Conversely, no amorphous phase was identified in samples A0, A1 and B0, though the δ 7 Li is shifted from the α max line. Thus, the precipitation of secondary clay minerals is not enough to explain the δ 7 Li shift and the role of other phase-like oxides must be considered.

Fe-Mn oxides, one of the main oxides in the non-residue part of soils and river sediments, have an exceptional tendency (particularly Mn oxides) for scavenging trace elements (Cu, Zn, Ni, Pb, etc.;[START_REF] Carpenter | Fe-Mn oxide coatings in stream sediment geochemical surveys[END_REF], Li not being investigated to our knowledge. One of the few studies describing Li-isotope values in Fe-Mn oxides is that of [START_REF] You | Precise determination of lithium isotopic composition in low concentration natural samples[END_REF], giving δ 7 Li values between +22.9 and +33.4‰ for hydrothermal ferromanganese crusts, such values being controlled by post-depositional exchange with seawater. To overcome this lack, we can use data from the labile fraction extracted from soils and river sediments in the same catchment [START_REF] Négrel | Investigating the sources of the labile fraction in sediments from silicatedrained rocks using trace elements, and strontium and lead isotopes[END_REF][START_REF] Négrel | Isotopic evidence of lead sources in Loire River sediment[END_REF]. These were obtained by leaching with cold and dilute acid, which releases the total inventory of non-residual trace elements, particularly those associated with hydrous Fe-Mn oxides adsorbed on clays, and occurring in carbonates and sulphides in the sediment load, as well as in natural organic matter. The content of the labile sample fractions from this study is consistently low, with similar ranges in soil and sediment (0.8-10.1% of total matter content; mean 6.4%). The Mn contents of this labile fraction range between 50 and 30,000 µg/g (mean ±6,000 µg/g). In addition, strong Li-Fe and Li-Mn correlations exist in agricultural soil at a European scale (GEMAS Project; Négrel et al., 2018). This is strongly related to precipitation of Fe-Mn oxide/hydroxide coatings, which provides a control over the lithium content within such phases that encourages investigating the labile fractions in soil and sediment for Li isotopes to evaluate Li isotope fractionation during the formation of Fe-Mn oxides.

-Conclusions

We carried out a systematic study of lithium-isotope compositions of surface-and groundwaters, and soil and sediment samples collected from a granite catchment in the French Massif

Central. This work incorporates many novel aspects, being an integrated investigation of weathering processes by means of lithium isotopes.

In water, the lithium isotope composition (δ 7 Li) of a dissolved load is generally higher than corresponding δ 7 Li values for groundwater, bedrock and sediment. It has been suggested

that Li-isotope fractionation in the dissolved load records silicate weathering intensity, as well as fluid-residence time, or exchange processes between water and sediment. In soil and sediment, Li is enriched through fractionation into the lithium-6 and -7 isotopes, leading to less negative δ 7 Li values and to changes in mineral abundance in the samples.

A model for determining the δ 7 Li of water after interaction with granite was applied, based on the assumption that Li derives from plagioclase, K-feldspar, apatite and biotite. The model calculations showed a relative agreement between the Li isotope composition predicted by the weathering model and that observed in water.

The fractionation process during granite weathering was investigated through noncongruent dissolution of the bedrock, leading to a δ 7 Li in solution. The main process caused by this solution is the fractionation of Li isotopes through the formation of secondary phases that preferentially incorporate light lithium ( 6 Li). This gives a residual solution with a specific δ 7 Li and another δ 7 Li value for the secondary phases.

We modelled weathering products with a fractionation factor that is consistent with the range of values found in the literature. Our results show that some, but not all, weathering products can be explained by such fractionation. Additional processes or inputs must therefore be investigated, such as the precipitation of another phase with a higher isotope fractionation rate, for which Fe-Mn oxides are a good candidate. Data from the Margeride were compared with those from the Strengbach granite catchment in the Vosges Mountains (NE France); again, the fractionation factor did not explain most Strengbach data and, here too, the role of another phase was suspected.

We highlighted the possible role of Li isotopes as tracers of water-rock interaction in a granite catchment. When evaluating regolith (soil and sediment) formation, any additional tracers to the basic ones (major-and trace elements, and Sr isotopes) are important for constraining the whole weathering system.

A remaining problem to be solved concerns the distinction between primary effects related to source/origin, and the effects of secondary processes (alteration, dissolution, precipitation) that can modify the isotopic signature, and are key reactions for fractionating Li isotopes. Another approach to understanding and interpreting the isotopic signatures will require Reactive Transport Model (RTM) computer simulations, to arrive at simultaneous solution of the governing equations for solute and geochemical transformations.

Figure 2 .

 2 Figure 2. Percentage change of (mobile) Li relative to (immobile) Ti (Equation1).

Figure 3 .

 3 Figure 3. Relationship between δ 7 Li values (‰) and Li contents (µg g -1 ) for bedrock, grit, surface saprolite (A0, B0), riverbank sediments (A1, B1) and soil (A2, B2). The separate minerals from the granite define the greyed area with the addition of residual minerals.Contrary to station D19, the samples from station D17 show more erratic variations.

Figure 5 .

 5 Figure 5. Plot of δ 7 Li versus the residual Cl concentration (μmol L -1 ) for surface water, once corrected from precipitation input (see text). The mean weighted δ 7 Li values for the fertilizers and Li additives used in agricultural practices are indicated.

Figure 6 .

 6 Figure 6. Relationship between the δ 7 Li and 87 Sr/ 86 Sr for Margeride granite, separate minerals and Mazel grit sample, and surface and mineralized spring waters. δ 7 Li and 87 Sr/ 86 Sr of the remaining trace minerals (titanite, rutile, epidote, zircon) are indicated in the graph (see text). The result of the weathering model is indicated by the grey circle (see text). The separate minerals from the granite define the greyed area with the addition of residual minerals.5.3.3 Modelling Li isotopic fractionation using a Raleigh equation: the role of secondary

Figure 7 .

 7 Figure 7. Li isotopic composition of surface saprolite (A0, B0), riverbank sediments (A1, B1) and soil samples (A2, B2) as a function of τLi (Equation1). Straight lines represent the evolution of δ 7 Li vs. τLi given by the batch equilibrium model according to Equation3.The Margeride data can be compared with other granite areas, but studies on granite and

Table 1

 1

	. Results of Li concentration (μg g -1 ) and δ 7 Li for parent rock, grit, separate minerals,
	sediment and soil, expressed in ppm. Results of Li concentration (μg g -1 ) and δ 7 Li for thermo-
	mineral waters (e.g., saline waters cold on surface, but linked to a deep hot reservoir) from

Acknowledgements

This work was financially supported by the BRGM Research Division 'Isotopes' research programmes, and benefited from collaboration by the BRGM Chemistry laboratories for major-and trace element analyses: T. Conte is thanked for his assistance, as well as M. Robert for her help in the Neptune laboratory. Financial support from the Région Centre is acknowledged for acquisition of the Neptune MC-ICP-MS. We are grateful to Dr. H.M. Kluijver for proofreading and editing the English text. Paul Tomascak, two anonymous reviewers and Editor-in-Chief Karen Johannesson are warmly thanked for

28 their critical comments and constructive reviews that greatly improved the manuscript, and for proofreading and editing the English text. This paper is dedicated to the memory of our wonderful colleague and friend, Dr. Tom Bullen, who recently passed away [START_REF] Shouakar-Stash | A remembrance of Thomas (Tom) Bullen, 1951-2018[END_REF]


Dr Célia M Silveira

Patrícia R Rodrigues

Dr Wissam Ghach

Dr Sofia A Pereira

Dr Francisco Esteves

Dr Michel Kranendonk

Mathieu Etienne

Prof. Dr Maria Gabriela Almeida
email: mg.almeida@fct.unl.pt

Dra Patrícia

Prof. Dr R Rodrigues

REQUIMTE Ucibio

Dr Etienne Mathieu

Electrochemical activity of cytochrome P450 1A2 -the relevance of O2 control and the natural electron donor

Keywords: Cytochrome P450, Cytochrome P450 oxidoreductase, CYP electrochemistry, O2 reduction, sol-gel

The direct electrochemical response of the membrane-bound human cytochrome P450 1A2 (CYP1A2) was studied on pyrolytic graphite electrodes, while encapsulated on a sol-gel matrix. The enzymatic reduction of O2 was evaluated in the presence and absence of its electron donor partner, cytochrome P450 oxidoreductase (CPR). When used without CPR, CYP1A2 was shown to be highly sensitive to O2 even in the presence of residual amounts.

Under aerobic conditions (air-saturated solutions) the catalytic signal due to the reaction with O2 was lost, suggesting the enzyme was inactivated. In contrast, the CYP1A2/CPR complex retained O2 reductase activity with high O2 concentration in solution. The results demonstrated a crucial role for CPR in stabilizing the immobilized CYP1A2 enzyme and in the preservation of O2 electrocatalysis, in this electrochemical set-up. Though the enzyme's monooxygenase activity towards caffeine was not detected, this study highlights the complexity in coupling CYP1A2 reduction currents with substrate turnover, due to the simultaneous electrochemical measurement of the O2 reduction reaction.

Introduction

Cytochromes P450 (CYPs) constitute a large family of b-type heme-containing enzymes belonging to the group of monooxygenases [1] that are present in both eukaryotic and prokaryotic organisms. They catalyze a wide variety of biological oxidation reactions, including the biosynthesis of hormones and of structural components of cells, as well as the metabolism of exogenous compounds. In mammals CYPs are membrane associated enzymes, either located on the inner membrane of mitochondria or on the cytoplasmic side of the endoplasmic reticulum (ER), where they play an important role in the biotransformation of xenobiotics and endobiotics. [2] In vivo, the heme iron utilizes two protons, O2 and two electrons, which derive from NADPH via a reductase enzyme, to oxidize organic substrates with stereo-and regio-selectivity. [2a] In the case of ER-located CYPs, electron donation occurs through the activity of cytochrome P450 oxidoreductase (CPR).

Because of CYPs versatility in i) recognition of different biological and xenobiotic compounds and ii) type of chemical reactions it can mediate, there is great interest in their application in biosensors [3] and in biocatalysis, [4] respectively. A major strategy of exploitation is through immobilization of the enzymes on electrodes that function as a controllable source of electrons to drive CYP catalytic activity. [5] However, interfacing these enzymes on electrode surfaces to sustain CYP's reaction cycle electrochemically has proven to be challenging. [6] In direct electron transfer (DET) based systems, CYP is wired to the interface, directly exchanging electrons with the electrode. [3a] Therefore, the enzyme needs to be immobilized in a manner that enables this exchange, but simultaneously does not hinder substrate binding or product release. The preservation of CYP's structural properties is a concern, particularly due to the relative ease with which it can be converted into the catalytically inactive cytochrome P420 conformation. [3a,6-7] Several approaches have been used for protein immobilization, either by direct attachment to electrodes, [8] binding in self-assembling monolayers, [9] immobilization in polymers and lipid membranes, [10] or the use of additives and binding-promotors, such as surfactants and nanomaterials. [6,11] Despite success in achieving DET and monitoring the Fe (III) + 1e -↔ Fe (II) reversible conversion, turnover is often low or absent, [4b,6,10c,10d,11a] which attests to the difficulties in reproducing the complex electron transfer (ET) processes in CYP catalytic activity. Aside from possible inactivation/denaturation of the enzyme due to the immobilization procedures, side reactions that consume electrons, but do not lead to substrate conversion, may confound data. [6][7]12] Reduction of O2 is at the basis of these uncoupling reactions that lead to the production of reactive oxygen species (Fig. S1). The auto-oxidation pathway involves the slow release of O2 -• , returning the heme iron to its ferric resting state (CYP-Fe(III)). In the peroxide shunt, the ferrous O2 bound complex, CYP-Fe(II)-O2, is reduced to yield H2O2. Finally, the oxidase shunt pathway bypasses substrate conversion steps after the formation of the CYP-(Fe(IV)=O) +• catalytic species by reducing it, releasing H2O and regenerating CYP-Fe(III). [13] The efficiency of the electrochemically driven catalytic reaction may be limited by these uncoupling pathways, which may also occur under physiological conditions. [13] Moreover, the formed reactive oxygen species may react with CYP either inactivating the enzyme or activating alternative reactions mechanisms. For example, the peroxide shunt pathway has been proposed as an electrochemical activation mechanism for CYPs, which is based on H2O2 reaction with CYP-Fe(III) to form the catalytic species CYP-(Fe(IV)=O) +• . [5,7a,10c] Another potential issue for the electrochemical response of CYPs, particularly in the development of biosensor devices, lies in the measurement of catalytic activity. The CYP's catalytic cycle involves binding and reduction of O2, as described above. But non-bound O2 is also electroactive in most potential windows used to monitor redox processes and therefore, it may be difficult to assign a cathodic current response to the electrocatalytic enzyme reaction. Consequently, measurement of products, following electrolysis reactions, has been used in various works to confirm substrate turnover. [6,14] This current report describes the study of the direct electrochemical response of human cytochrome P450 1A2 (CYP1A2) alone or in combination with its oxidoreductase (CYP1A2/CPR). The proteins were immobilized in a hybrid sol-gel thin film, based on a water-based sodium silicate, and polyethylene glycol (PEG). [15] The DET reaction of the enzyme was characterized by cyclic voltammetry in the absence and in the presence of CYP1A2 substrate, caffeine, and with different concentrations of the co-substrate, O2. The effects of O2 on CYP's stability and catalytic response, as well as, the experimental issues encountered during the evaluation of the bioelectrochemical system are discussed.

Experimental

Chemicals

Sodium silicate solution (14 % NaOH, 27 % SiO2), caffeine, glucose, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP + ), and resorufin were acquired from Sigma-Aldrich. Polyethylene glycol (PEG) 400 was purchased from Prolabo and hydrochloric acid solution was from Riedel-de Haën.

Ethoxy-resorufin was obtained from BD Biosciences. Phosphate buffers were prepared from 0.5 M stock solutions of NaH2PO4.2H2O (Prolabo) and Na2HPO4.2H2O (Fluka). All solutions were prepared with deionized water (18 MΩ cm) obtained from a Millipore MilliQ water purification system.

Enzymes

Membrane-bound recombinant human CYP1A2 (1.22 µM in 75 mM Tris-HCl, 10% (v/v) glycerol and 25 mM EDTA, pH 7.5) was obtained by expression in E.coli strain DH5α.

Culturing, microsome preparations and CYP-content determinations were performed as previously described. [16] Recombinant CYP1A2/CPR membrane-bound complex (coexpressed in E.coli, 1:1 stoichiometry) was acquired from Cypex and used as provided (1 µM CYP1A2, in 50 mM Tris-acetate buffer, pH 7.6, 250 mM sucrose and 0.25 mM EDTA), except for the ethoxy-resorufin dealkylation activity measurements of CYP1A2 for which a different recombinant preparation was used (see section 2.5). Catalase [Cat] (bovine liver, 2000 -5000 U mg -1 ) and glucose oxidase [GOx] (Type II Aspergillus niger, 19.3 U mg -1 ) were purchased as lyophilized powders from Sigma-Aldrich and prepared in 0.05 M phosphate buffer, pH 7.0. Cat and GOx were used in the O2 scavenging system at final concentrations of 2000 and 15 U mL -1 , respectively. Cat was used in the H2O2 scavenging system also at 2000 U mL -1 .

Bioelectrode preparation

The working electrodes (3 mm diameter disks) were made of basal plane pyrolytic graphite

[PG] (GE Healthcare). The surfaces were treated by abrasion with sandpaper and polished with alumina slurry (0.3 µm, Buehler), followed by sonication in deionized water. Finally, the electrodes were washed with deionized water and dried with compressed air.

The hybrid sol-gel was prepared as previously described. [15] The starting sol solution contained 55 mM sodium silicate and 6 % PEG 400; this mixture was thoroughly mixed and adjusted for pH 7.0 with 1 M HCl.

Electrodes were modified by placing a 7 μL drop of a 1:1 mixture containing membrane preparations of CYP1A2 or CYP1A2/CPR complex (each with 1 μM CYP1A2) and sol-gel onto the clean surface. Electrodes were then air dried for 1 h. Control experiments were performed with sol-gel modified electrodes (without CYP1A2), and with CYP1A2 simply adsorbed on a clean electrode surface or immobilized in 1:1 mixtures with either 55 mM sodium silicate or 6 % PEG 400.

Electrochemical measurements

A conventional three-electrode electrochemical cell was used with an Ag/AgCl reference electrode, a Pt counter electrode (both from Radiometer) and the enzyme/sol-gel modified PG electrode. The electrochemical cell contained 10 mL of 0.05 M phosphate buffer, pH 7.0, as supporting electrolyte. Unless stated otherwise, the cell was maintained under an argon atmosphere by thoroughly purging the solution before the experiments (20 min -1 h) and maintaining the argon flow on top during measurements. Assays in the presence of O2 were performed with air-saturated supporting electrolyte. For non-catalytic assays, an O2 scavenging system composed of GOx, Cat and glucose was added to the cell previously purged with argon.

Measurements were performed using a potentiostat Autolab PSTAT 12 (Eco-Chemie), using the NOVA 1.6 software (Eco-Chemie) for control and data acquisition. In the cyclic voltammetry experiments, a 50 mV s -1 scan rate was used, unless stated otherwise. The catalytic response was tested by adding small volumes (0.1 -0.4 mL) of argon-purged or airsaturated water and caffeine (stock solution 60 mM) to the electrochemical cell, as detailed in the results section. The supporting electrolyte was magnetically stirred after each injection.

Final caffeine concentrations in the cell ranged from 0.6 to 4.8 mM. A Clark-type O2 electrode (Tekcoplus) was used to monitor the dissolved O2 concentrations in the solutions added to the electrochemical cell and in the supporting electrolyte prior to starting the assays.

All potentials are quoted against the Ag/AgCl reference electrode (+207 mV vs SHE).

CYP1A2-mediated ethoxy-resorufin dealkylation assays

The catalytic activity of CYP1A2 [EthR-O-deethylation, EROD] in presence or absence of sol-gel, was evaluated using membrane fractions isolated from Escherichia coli cell model BTC (heterologous co-expression of membrane-bound human CYP1A2 together with human full-length CPR), as reported previously. [17] A range of EthR concentrations (0 -5 µM) and 8 nM CYP1A2 (with or without an equal volume of sol-gel solution) were used. Initial reaction rates were measured in triplicate. The Michaelis-Menten equation was fitted to the plots of initial reaction rate vs substrate concentration (r 2 > 0.95) allowing the determination of steady-state kinetic parameters, using GraphPad Prism 5.01 Software.

Results and Discussion

Immobilization of CYP1A2 in a sol-gel film

The direct electrochemical response of CYP1A2 was evaluated on PG electrodes upon immobilization in a sodium silicate/PEG 400 based sol-gel. [15] Cyclic voltammograms (CVs) obtained in argon-purged solutions reveal a broad and intense cathodic wave centered at -0.335 V (Fig. 1D). In contrast, only small reduction peaks (between -0.3 to -0.5 V) are observed when CYP1A2 is directly adsorbed on the bare PG, or immobilized on either a PEG 400 or a sodium silicate film, respectively (Fig. 1A-C). This demonstrates that the hybridization of the sol-gel film with the organic polymer, PEG 400, enables the immobilization of the protein in an electrochemically active form, with a sufficient fraction properly orientated towards the electrode. The use of PEG 400, in particular, has been described as highly beneficial for the stabilization of membrane proteins. [15] The signal decreases rapidly in the first minutes after introduction of the CYP1A2/sol-gel/PG electrodes in the electrochemical cell, but stabilizes after approximately 20 cycles. This signal decay is reproducible and may be attributed to the reduction of traces of O2 in the electrochemical cell by the encapsulated CYP1A2. [11b] Further evidence of O2 catalytic reduction is the absence of anodic signal, which is a characteristic voltammetric signature of electrochemical catalysis. [18] Note that the cathodic signal decrease is also observed with the sodium silicate and PEG 400 films, but is significantly less pronounced in the protein-only films, which is likely to retain considerably less O2 (Fig. 1A). Results obtained with sol-gel control electrode (without CYP1A2) confirm the presence of small amounts of O2 in the argon-purged electrochemical cell and its permeation through the sol-gel film, as the direct reduction of dissolved O2 at the electrode surface is observed below -0.5 V (Fig. S2A).

Therefore, we conclude that under the applied experimental conditions, and due to the high affinity of the ferrous heme to O2, the electrochemical response of CYP1A2 in the sol-gel electrodes was dominated by the catalytic reduction of O2.

Direct electrochemistry of CYP1A2 in the presence of a O2 scavenging system

Cathodic processes are often subject to interference from dissolved O2, since it can be reduced directly at the electrode surface or react with enzymes, analytes or electron donors (e.g. redox mediators, reducing agents) involved in the electrochemical reaction. To avoid the resulting background currents, O2 is typically removed from the electrolyte solution by purging it with an inert gas before measurements. Alternatively, different chemical and enzyme-based O2 scavenging systems have been explored for maintaining anaerobic conditions (often in open systems) without the need for laboratory-based equipment. [19] To further characterize the electrochemical response of the CYP1A2/sol-gel electrodes we employed the GOx/Cat system in order to study the direct electrochemical response of the CYP1A2/sol-gel electrodes without interference of residual O2. The system is based on the catalytic oxidation of glucose by GOx with concomitant reduction of O2 (eq. S1); the H2O2 generated from this reaction is dismutated into H2O and O2 by Cat (eq. S2). The resulting O2 is further reduced by GOx. In the net cyclic reaction, two glucose molecules are used for the reduction of one O2 molecule (eq. S3). The high turnover rates of the two enzymes leads to a rapid decrease of O2 content in solution. [19] Upon addition of the enzymatic O2 scavenging system to the argon-purged electrolyte solution, the cathodic currents assigned to the direct reduction of O2 at the sol-gel control electrode (without CYP1A2), are quickly eliminated (within 3 minutes, Fig. S2A). This indicates that even after continued purging with argon (up to 1 h), traces of O2 can remain in the electrochemical cell and diffuse through the sol-gel film. The two enzymes can thus remove the residual O2 more efficiently than the physical purging with argon. A reversible electrochemical signal, centered at -0.41 V, is observed in the CVs after prolonged incubation with the O2 scavenging system (20 min) (Fig. 2SB). This was attributed to the system components, possibly the free flavin cofactor, a common impurity in commercial GOx preparations, and suggests the small cofactor may be adsorbing to the sol-gel film. [20] The CVs of the CYP1A2/sol-gel/PG electrodes obtained in the presence of GOx, Cat and glucose, revealed two current peaks (Fig. 2) in addition to the signals of the O2 scavenging system, previously observed with the control electrode. The peaks can be assigned to CYP's Fe(III)/Fe(II) conversion. The anodic and cathodic peak currents are identical and proportional to the scan rates, while peak separations vary between 30 and 90 mV in the range of scan rates investigated. These characteristics are consistent with a quasi-reversible diffusion-less electrochemical process. The formal reduction potential, -301±17 mV vs Ag/AgCl, is constant over the whole scan rate range and in agreement with the reported value for CYP1A2 immobilized in polystyrene sulfonate films. [18] Measurements were performed in 0.05 M phosphate buffer, pH 7.0, purged with argon and upon addition of GOx (15 U mL -1 ), Cat (2000 U mL -1 ) and glucose (50 mM).

The results obtained with the argon-purged supporting electrolyte versus the additional use of the enzyme-based O2 scavenging system, confirm that the electrochemical activity with the immobilized CYP1A2 is highly sensitive to the presence of O2. In contrast to argon purging alone, addition of GOx and Cat can remove trace amounts of O2, probably trapped in the enzyme film, revealing the direct electrochemical response of the sol-gel immobilized CYP1A2 free from O2 interference.

Electrocatalytic activity of CYP1A2

The O2 dependence of CYP1A2/sol-gel/PG electrochemical response is depicted on Fig. 3A.

The experiment was initiated with argon-purged electrolyte solution to which small volumes of air-saturated water (O2 7.23 ppm/0.226 mM) were added, corresponding to an increase in O2 concentration up to 17.4 µM. Monitoring of the O2 content in the solutions injected into the electrochemical cell was performed with a Clark-type dissolved O2 electrode. CYP1A2's peak currents increased proportionally with added water volumes, as a result of the electrocatalytic reduction of the added O2 (Fig. S3). However, when the electrode was exposed to a higher concentration of O2 namely, to air-saturated electrolyte (approximately 0.22 mM O2), the cathodic signal, although intense in the first scan, quickly disappeared during subsequent continuous cycling (Fig. 3B). The peak shifted to lower potentials (approximately -0.7 V), which is consistent with direct reduction of O2 on the PG electrode. Although loss of activity in oxygenated solutions was previously reported for both human (CYP3A4) and bacterial (CYP101, CYPcam) enzymes, [4b,10c,21] the detrimental effects of O2 on CYP's electrocatalytic activity have rarely been discussed. Our results show that under relatively high O2 concentrations CYP1A2 electrochemical signal was lost, thereby compromising the measurement of its monooxygenase activity. Looking for possible explanations, we have investigated the previously proposed electrochemical activation pathways for CYPs, represented in Fig. 4. [5,10c] Adapted from [5,10d] .

In a DET based mechanism, the electrode replaces electron delivery by the CPR component.

Thus, after the one-electron reduction of CYP-Fe(III) on the electrode (step 1) and binding of O2 to form CYP-Fe(II)-O2 complexes (step 2), a second electron is transferred generating CYP-Fe(II)-O2 ˗• (step 3). Two subsequent protonation steps lead to the formation of the CYP-(Fe(IV)=O) +• catalytic species, responsible for substrate turnover. An alternative pathway for DET based biocatalysis under aerobic conditions was proposed by Rusling and co-workers [5,10c] . This pathway (see Fig. 4, dash-dotted lines) is known as the electrochemical peroxide shunt and is dependent on CYP activation by H2O2 formed by the electrocatalytic reduction of O2. This reduction (step 8) is supported by an observed sharp increase in cathodic current in the presence of minute concentrations of O2 (cf section 3.1). H2O2 may then react with CYP-Fe(III) to form the catalytic intermediary CYP-(Fe(IV)=O) +• (step 9); this species can also be reduced at the electrode to regenerate CYP-Fe(III) (step 10). [5,10c] We propose that in the presence of elevated O2 levels, the cycle is blocked at some point. For this blockage, we suggest the following hypotheses: i) the conformational change required to convert CYP-Fe(II)-O2 into H2O2 and CYP-Fe(III)/(II) recycling [22] does not occur (blocking of step 8); ii) the accumulation of high amounts of H2O2 and/or the excessive cathodic current due to the reduction of O2 to H2O2 leads to CYP degradation and to the consequential interruption of the cycle in step 9. To test the latter hypothesis, the electrochemical assay was performed in the presence of Cat, a H2O2 scavenging enzyme. No relevant differences were detected in the CVs in air-saturated solutions in the presence or absence of 2000 U.mL -1 Cat, suggesting that the loss of activity of the immobilized CYP1A2 is not caused by the accumulation of H2O2 in the electrochemical cell (Fig. S4). However, since Cat operates in solution we cannot rule out the possibility that a high local concentration of H2O2 can be generated inside the sol-gel film, before diffusion to the bulk solution, which could damage the immobilized CYP1A2.

To test the 1 st hypothesis, O2 reduction was evaluated in the presence of CYP1A2's electron donor protein, CPR. These results are presented and discussed in detail on section 3.4. The overall conclusion is that maintenance of catalytic O2 reduction by CYP1A2 is favored in the presence of CPR. Accordingly, we propose that CPR is essential for the maintenance of the peroxide shunt pathway, avoiding a potential blockage during the conversion of CYP-Fe(II)-O2 into H2O2 and CYP-Fe(III)/(II) (step 8 in Fig. 4).

The catalytic activity of the CYP1A2/sol-gel hybrid films towards caffeine was subsequently studied. As detailed above, air-saturated solutions cannot be used, as the protein signal is lost in these conditions. Consequently, the experiment was initiated in argon-purged supporting electrolyte and small volumes of air-saturated caffeine solution (O2 content 6.68 ppm/0.209 mM) were added to the cell (0.6 -4.8 mM caffeine and 2.1 -16.1 µM O2 final concentrations, Fig. S5). Note that, we used caffeine concentrations above the reported KM value in solution (240 -280 µM range) [23] to ensure the enzyme worked at maximum rate, thus facilitating detection of electrocatalytic activity. The cathodic peak intensity increased with caffeine concentration, however the current increase was identical to the one obtained by adding equivalent volumes of air-saturated water (Fig. S3). This suggests that the observed response was due to the catalytic reduction of O2 and not caffeine turnover. In the control experiments, performed with argon-purged caffeine solutions, no catalytic activity was observed (Fig. S6), despite the presence of O2 traces. Therefore, we conclude that the catalytically active state leading to substrate conversion was not formed under these experimental conditions.

Uncoupling reactions occur frequently with mammalian CYPs; they yield O2 reduction species but do not drive product formation. [6,10d] Besides the peroxide shunt pathway and the production of H2O2, the electrochemical set-up may also favor the occurrence of the oxidase shunt pathway. The potentials applied may have been sufficiently low to regenerate the resting state species (CYP-Fe(III)) without substrate conversion. In other words, the catalytic species CYP-(Fe(IV)=O) +• would be rapidly reduced to CYP-Fe(III)/(II), increasing the flow through steps 10 and 1 and bypassing product formation (steps 6 and 7 in Fig. 4). In contrast to the on/off binding of CYP's redox partner CPR, the continuous electric fields generated at the electrode surface could explain the inability to form CYP-(Fe(IV)=O) +• . Also, the natural occurring intermittent attachment/detachment of CPR, with the involvement of CPR's extensive open/close protein dynamics, could induce conformational changes in CYP, which might be of relevance for the reaction pathway in substrate conversion. [24] Overall, our results recommend caution when evaluating an increase of cathodic current in the presence of O2 and organic substrates. This has been used as a measure for catalytic bioconversion in some studies reporting on the electrocatalytic activity of electrode immobilized CYPs towards organic substrates. [11d,25] Care must be taken to guarantee that current increase is not mistaken by a higher rate of O2 reduction, occurring when substrate is added to the cell, an issue that has not been suitably addressed so far. Electrochemically driven CYP biocatalysis studies must be looked at with caution and should include appropriate control experiments, to provide reliable results of protein activity. [26] These include the control of O2 concentration, the measurement of current upon solvent addition, as well as the detection of reaction products, a strategy previously used by several authors, but that is not always applicable. [9,10c,14,17,27]

Direct electrochemistry of CYP1A2/CPR complex

As mentioned above, the hypothesis that an ET-coupled conformational change is required for O2 reduction and regeneration of CYP-Fe(III)/(II) was evaluated in the presence of CPR.

In the natural catalytic cycle of CYPs, the electron donor protein, CPR, forms a complex with CYP and is responsible for the electron transfer from NADPH to the enzyme. [5] The CYP1A2/CPR complex behaves similarly to the CYP1A2 electrodes in argon-purged supporting electrolyte and in the presence of the enzymatic O2 scavenging system. In the first case, a cathodic peak, assigned to O2 catalytic reduction, is detected at -0.320 V vs Ag/AgCl.

Then, upon addition of GOx, Cat and glucose to the electrochemical cell, the catalytic currents are eliminated and CYP's Fe(III)/Fe(II) reversible reaction is observed at a potential of -0.314±8 V vs Ag/AgCl (Fig. S7). The low intensity peak pair, centered at -0.42 V vs Ag/AgCl was initially attributed to the components of the enzymatic O2 scavenging system, as described in section 3.2 (cf. Fig. 2A). However, the control experiments performed with CPR alone, i.e. upon immobilization of CPR-containing membrane fragments in the sol-gel matrix (Fig. S8), showed that this signal was already present before the addition of the scavenging system (Fig. S8, solid black line). Therefore, the coinciding signal was here assigned to one of CPR's redox centers. By comparison with the electrochemical response of other CYPs, the signal observed at -0.42 V was attributed to the FAD center. Also note that the FMN cofactors typically have higher reduction potentials than the FAD and heme cofactors. [10d,28] In air-saturated supporting electrolyte (O2 approximately. 0.22 mM), CVs of CYP1A2/CPR/sol-gel PG electrodes revealed a sharp catalytic peak at a similar potential observed for CYP1A2/sol-gel system (-0.350 V and -0.400 V, respectively), Fig. 5. However, in the presence of CPR, the cathodic peak corresponding to O2 catalytic reduction was maintained after an initial decrease. Upon stirring the solution, the O2 concentration at the electrode interface was equilibrated with the bulk solution and the signal intensity was restored to values only slightly lower than the initial current. This indicates that unlike the CYP1A2/sol-gel electrodes (Fig. 3B), CYP1A2/CPR complex remains active towards O2 reduction in the presence of high concentrations of this molecule. As already noted, in these conditions the conformational changes required to recycle CYP-Fe(III)/(II) may not occur, possibly because the electrode surface cannot mimic the interaction with CPR. Nonetheless, no caffeine related catalytic currents could be detected with the CYP1A2/CPR/sol-gel electrodes in the presence of either argon-purged or air-saturated caffeine solutions (not shown). The current variations were identical to those obtained upon injection of equal volumes of solvent, suggesting a direct correlation with O2 reduction by the immobilized CYP1A2, as observed with the CYP1A2/sol-gel electrodes. Importantly, we observed a consistent positive shift of the catalytic O2 reduction peak between CYP1A2 and CYP1A2/CPR electrodes (Fig. 6). This higher reduction potential suggests a facilitated catalytic response of CYP1A2 in the presence of CPR.

Taken together, our results indicate that CPR has a critical role in the stabilization of CYP1A2 and in the maintenance of O2 electrocatalysis. Nevertheless, the proposed sol-gel formulation does not ensure the preservation of the immobilized enzyme's activity towards caffeine. In order to understand the reasons behind the lack of activity, we investigated the spectroscopic features of CYP1A2 in dried sol-gel films, which enable the identification of inactive CYP states, such as the P420 configuration. Unfortunately, all our attempts to characterize the enzyme/sol-gel films either by UV-Vis or resonance Raman spectroscopy, were thwarted by the low amount of immobilized sample and the complexity of microsome preparations (not shown). [6][7] Next, to rule out any possible influence from the sol-gel matrix on the CYP1A2/CPR activity, we checked the catalytic response, in the presence of the sol-gel, through a solution enzyme assay specific for CYP1A2. [17] As shown in Fig. S9, the turnover rate obtained for the enzyme/sol-gel mixtures was only slightly lower (kcat app 0.36 s -1 ) than the value for the enzyme alone (kcat 0.49 s -1 ), which confirms that the matrix provides a friendly environment for the enzyme. Since the presence of sol-gel per se does not significantly impair the enzyme activity in solution, CYP1A2 immobilization on the electrode surface may result in steric hindrance, like an improper orientation on the electrode that blocks the substrate access to active site.

Conclusions

Herein we report on the DET of a hybrid CYP1A2/sol-gel film on pyrolytic graphite electrodes. Our results showed that the synergic combination of sodium silicate and PEG 400 provided a friendly environment for the enzyme immobilization in an electrochemically active form. Also, we demonstrated that a GOx/Cat-based O2 scavenging system was able to efficiently remove O2 from the sol-gel film, contrary to the commonly used argon purging method. A non-turnover response from the immobilized CYP1A2, without O2 interference, could only be attained in the presence of this O2 scavenging system. Furthermore, this work proves that the measurement of CYP electrochemical activities in the presence of O2 is not straightforward. Control assays concerning the regulation of the cosubstrate's concentration are mandatory in studies aiming to correlate CYP catalysis with current variations, as they are essential to correctly assign catalytic currents. In particular, the addition of O2-containing substrate solutions to the electrochemical cell should be carefully monitored.

Despite the absence of monooxygenase activity, the immobilization of CYP1A2 in the presence of its physiological electron transfer partner CPR (i.e. the CYP1A2/CPR complex) was shown to be fundamental for the maintenance of O2 electrocatalysis. Reduction of O2 in air-saturated solutions could be measured with the CYP1A2/CPR complex/sol-gel electrode but not with the CYP1A2/sol-gel system.

Fig. 1 .

 1 Fig. 1. Cyclic voltammetry response of CYP1A2, either (A) directly adsorbed on the PG

Fig. 2 .

 2 Fig. 2. (A) Cyclic voltammetry response of the control sol-gel/PG (dotted line) and

Fig. 3 .

 3 Fig. 3. Cyclic voltammetry response of CYP1A2/sol-gel PG electrodes in the presence of O2.

Fig. 4 .

 4 Fig. 4. Proposed CYP catalytic cycle indicating the peroxide shunt electrochemical activation

Fig. 5 .

 5 Fig. 5. Cyclic voltammetry response of CYP1A2/CPR/sol-gel electrodes in air-saturated

Fig. 6 .

 6 Fig. 6. Cyclic voltammetry response of (a) CYP1A2/sol-gel and (b) CYP1A2/CPR/sol-gel

Acknowledgements

The authors acknowledge the support of the Erasmus program from the European Union. This work was supported by national funds from FCT -Foundation for Science and Technology, I.P. through the CiiEM (project IDB/04585/2020), and the Applied Molecular Biosciences Unit-UCIBIO (project UID/Multi/04378/2013), co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). CMS thanks the financial support from Fundação para a Ciência e Tecnologia (Fellowship SFRH/BPD/79566/2011) and from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular), which is funded by FEDER funds through COMPETE 2020-Programa Operacional Competitividade e Internacionalização (POCI). MK and FE were partly supported by grant UID/BIM/00009/2019 (ToxOmics) from Fundação para a Ciência e a Tecnologia.

Cytochrome P450 1A2 (CYP1A2) direct electrochemistry was obtained in a biocompatible sol-gel film.

Control of O2 levels is critical to get reliable results on the enzyme's activity.

The electrocatalytic activity towards O2 depends on interaction with the electron donor CYP oxidoreductase (CPR).