Alessandra Flores

Jorge Ramirez-Franco

Richard Desplantes

Kévin Debreux

Géraldine Ferracci

Florian Wernert

Marie-Pierre Blanchard

Yves Maulet

Fahamoe Youssouf

Marion Sangiardi

Cécile Iborra

Michel Robert Popoff

Michael Seagar

Jacques Fantini

Christian Lévêque

Oussama El Far
email: oussama.el-far@inserm.fr.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B

Keywords: Botulinum neurotoxin B receptor, synaptotagmin, gangliosides SIGNIFICANCE

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

INTRODUCTION

Botulinum neurotoxins (BoNTs) reach their neuronal targets through several steps.

After oral ingestion, they bind to epithelial cells of the digestive tract, then escape from the gastro-intestinal milieu, mainly by trancytosis and barrier disruption, into the circulation (1) [START_REF] Simpson | The life history of a botulinum toxin molecule[END_REF]. BoNTs then cross the endothelial barrier and attain peripheral nerve terminals, binding with high affinity to specific neuronal receptors. BoNT serotypes A, B and E (BoNT/A, BoNT/B, BoNT/E), the main agents of human botulism, target mainly neuromuscular junctions (NMJ), where they impair acetylcholine release and consequently cause flaccid paralysis [START_REF] Tighe | Botulinum neurotoxins: mechanism of action[END_REF].

At the molecular level, BoNTs are AB toxins composed of two polypeptides, the heavy (100 kDa) and the light (50 kDa) chains, linked by a disulphide bridge and noncovalent interactions [START_REF] Poulain | Why Are Botulinum Neurotoxin-Producing Bacteria So Diverse and Botulinum Neurotoxins So Toxic?[END_REF]4,[START_REF] Kammerer | Botulinum neurotoxins: new questions arising from structural biology[END_REF]. All BoNT serotypes employ a similar mechanism of intoxication. The carboxy and amino-terminal domains of the heavy chain confer respectively receptor-binding and light chain translocation through the endocytic vesicle membrane into the cytosol. The light chain is a zinc-dependent endopeptidase responsible for the inactivation of SNARE proteins essential for neurotransmitter release. BoNT/B cleaves synaptobrevin (VAMP), which is an integral protein of the synaptic vesicle membrane, whereas BoNT/A and BoNT/E cleave SNAP-25 (synaptosomal-associated protein of 25kDa), a protein located on the cytosolic face of the presynaptic membrane (1) (2) (3).

BoNT/A, B and E receptors are composed of plasma membrane gangliosides and intra-luminal domains of transmembrane synaptic vesicle proteins that become accessible at the presynaptic membrane upon exocytosis. BoNT/A and E bind to the synaptic vesicle protein 2 (SV2) while BoNT/B binds synaptotagmin (SYT isoforms 1 and 2).

Gangliosides are amphiphilic glycosphingolipid molecules predominantly anchored, via their ceramide group, in the outer leaflet of plasma membranes. Their extracellular polar part is composed of several characteristic sugar molecules with the negatively charged sialic acids at precise positions. Gangliosides are major components of neuronal membranes accounting for 10-20% of total membrane lipids of the outer leaflet and act as cell surface receptors for certain viruses, bacteria, microbial toxins and antibodies [START_REF] Fewou | The pre-synaptic motor nerve terminal as a site for antibody-mediated neurotoxicity in autoimmune neuropathies and synaptopathies[END_REF][START_REF] Salinas | A hitchhiker's guide to the nervous system: the complex journey of viruses and toxins[END_REF]. GM1, GD1a, GD1b and GT1b account for >90% of adult mammalian brain gangliosides. They differ in the number and position of their sialic acids linked to a common tetra-saccharide core [START_REF] Fantini | Brain Lipids in Synaptic Function and Neurological Disease : Clues to Innovative Therapeutic Strategies for Brain Disorders[END_REF][START_REF] Schnaar | Gangliosides of the Vertebrate Nervous System[END_REF] with GT1b being the preferred form recognized by BoNT/B [START_REF] Chai | Structural basis of cell surface receptor recognition by botulinum neurotoxin B[END_REF]. Because of their high content in long saturated alkyl chains and their propensity to associate with other sphingolipids and cholesterol, gangliosides are thought to promote the formation of tightly packed lipid domains that are in a dynamic equilibrium with less ordered environment [START_REF] Montecucco | Presynaptic receptor arrays for clostridial neurotoxins[END_REF].

Moreover, the hydrogen bonding ability of their headgroups, results in extensive ganglioside and protein-ganglioside interactions in cis or trans configuration [START_REF] Sonnino | Membrane lipid domains in the nervous system[END_REF] [START_REF] Schnaar | Gangliosides of the Vertebrate Nervous System[END_REF].

In neurons, gangliosides are involved in a diverse range of functions including synaptic transmission [START_REF] Schnaar | Gangliosides of the Vertebrate Nervous System[END_REF] and co-localize with proteins having major role in neurotransmission such as SNARE proteins [START_REF] Sonnino | Membrane lipid domains in the nervous system[END_REF][START_REF] Chamberlain | SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis[END_REF].

SYT 1 and 2 are expressed in synaptic vesicles and plays an essential function in neurotransmitter release acting as the primary calcium sensors that trigger exocytosis. SYT also plays a major role in clathrin-dependent endocytosis in the periactive zone [START_REF] Yao | Uncoupling the roles of synaptotagmin I during endo-and exocytosis of synaptic vesicles[END_REF]. They consist of a small luminal N-terminal segment that harbors Nand O-glycosylation sites [START_REF] Kwon | Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV2 and synaptophysin to recycling synaptic vesicles[END_REF], a single transmembrane domain (TM), and a large cytoplasmic calcium, phospholipid and SNARE complex binding domain participating in membrane bilayer deformation necessary for SV fusion [START_REF] Chapman | A Ca(2+) Sensor for Exocytosis[END_REF]. Upon exocytosis the N-terminal domain of SYT becomes accessible at the cell surface. Although there are minor sequence differences between SYT1 and SYT2 N-terminal domains, the affinity of BoNT/B for rat SYT2 is more than two orders of magnitude higher than for rat SYT1 [START_REF] Jin | Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity[END_REF]. Interestingly however, a single amino acid difference between rat and human SYT2 drastically diminishes BoNT/B binding affinity for the latter and consequently SYT1 is considered to be the major receptor for BoNT/B in humans at NMJ [START_REF] Peng | Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins[END_REF] and autonomic nerve terminals [START_REF] Elliott | Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models[END_REF].

X-ray crystallography has shown that the trefoil C-terminal domain of BoNT/B carries two vicinal but independent binding pockets. One pocket accommodates the polar part of a ganglioside, while the other interacts mainly with hydrophobic amino acids in the extracellular juxtamembrane domain of SYT [START_REF] Elliott | Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models[END_REF][START_REF] Berntsson | Structure of dual receptor binding to botulinum neurotoxin B[END_REF]. Although this SYT domain is unstructured in solution it has an α-helical structure in the toxin binding pocket [START_REF] Chai | Structural basis of cell surface receptor recognition by botulinum neurotoxin B[END_REF][START_REF] Jin | Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity[END_REF][START_REF] Berntsson | Structure of dual receptor binding to botulinum neurotoxin B[END_REF]. Structural modeling data and substitution of several amino acids between SYT1 and SYT2 have suggested a conserved BoNT/B binding mode for both SYT isoforms [START_REF] Jin | Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity[END_REF][START_REF] Elliott | Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models[END_REF].

Although structural, biochemical and functional studies support the idea that a double receptor anchorage of BoNT/B is responsible for the exquisite neurotropic specificity of BoNT/B, the precise sequence of events that lead to BoNT/B binding is unknown.

The current "dual receptor" hypothesis proposes that circulating BoNTs are initially captured by gangliosides and thus concentrated at the presynaptic surface. The GT1b/BoNT complex would then migrate in the plane of the membrane, facilitating subsequent binding to the protein receptor, making the interaction almost irreversible and allowing receptor-mediated endocytosis [START_REF] Montecucco | Presynaptic receptor arrays for clostridial neurotoxins[END_REF][START_REF] Montecucco | How do tetanus and botulinum toxins bind to neuronal membranes?[END_REF]. However, it was proposed that the toxin simultaneously binds both receptors located in very close proximity [START_REF] Schiavo | Structural biology: dangerous liaisons on neurons[END_REF]. This alternative view is moreover supported by the very low binding affinity of BoNT to gangliosides compared to SYT [START_REF] Stern | A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency[END_REF]. Recently a new partner has been implicated in the mechanisms that orchestrate BoNT/B binding to neurons, as membrane lipids have recently been shown to interact with a hydrophobic loop of BoNT/B located between the SYT and the GT1b binding pockets [START_REF] Stern | A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency[END_REF].

In this report we reexamined the interaction between BoNT/B and its co-receptors.

Using a range of molecular approaches, we first identified a consensus glycosphingolipid binding motif in SYT and then characterized a direct interaction between its juxtamembrane extracellular domain and the polar moiety of GT1b. Our results indicate that this interaction extends into the membrane, involving the TM region of SYT and the ceramide moiety, corroborating the view that the SYT/GT1b complex forms a highly stable structure, constituting a preassembled high affinity BoNT/B receptor. Our data suggest that GT1b binding confers α-helical structure to the extracellular juxtamembrane domain of SYT which is then accommodated in the SYT binding pocket of BoNT/B, while subsequent occupancy of the ganglioside binding pocket would then stabilize BoNT/B binding.

Results

Assessment of BoNT/B binding to SYT2 or GT1b in a cellular context. The current model for BoNT/B intoxication proposes that BoNT/B first attaches to the membrane by binding to gangliosides and then moves laterally to bind SYT. We revisited this question by investigating BoNT/B binding to SYT2 or GT1b separately.

These experiments were performed in SYT1 and 2-deficient PC12 cells transfected with SYT2 (PC12ΔSYT1 SYT2 + ) and treated with PPMP, an inhibitor of GT1b synthesis that abolishes most of its expression (98 ± 2%, n = 3 independent experiments, Fig. S1A andB). BoNT/B binding to PC12 cells was not detectable, neither with endogenous nor exogenously added GT1b, in the absence of SYT (Fig. S1A).

Moderate but specific BoNT/B binding was detected on PC12 cells expressing SYT2 and treated with PPMP. Staining was mainly detected at the cell surface, co-localized with SYT2 and delineated transfected cells (Fig. 1A) whereas no signal was observed on cells transfected with carrier vector expressing EGFP (lower lane). In the presence of PPMP and upon increasing BoNT/B concentration from 5 to 40 nM, BoNT/B binding was enhanced by a factor of 3 (Fig. 1A andB). The binding affinity of BoNT/B for SYT2 in the absence of GT1b was measured using SYT2-expressing exosomes that do not contain GT1b or GD1a [START_REF] Desplantes | Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor[END_REF] and yielded a K D of 40 nM (Table 1 and Fig. S1C). As a negative binding control, BoNT/E which binds SV2, did not show any interaction in this paradigm (Fig. S1C). These results indicate that while BoNT/B binds specifically to SYT2 in the absence of GT1b, binding to GT1b alone was not detectable.

In order to study on living cell membranes the potential proximity of SYT2 and GT1b co-receptors, close enough to capture BoNT/B, we used SYT2-transfected PC12 cells maintained on ice to reduce membrane dynamics and receptor diffusion. We quantified BoNT/B binding using a sensitive cell-based colorimetric assay in the absence or presence of PPMP. Under these conditions, BoNT/B specifically bound to SYT2-transfected cells (Fig. 1C). This binding was totally abolished upon preincubation of BoNT/B with a neutralizing antibody used to block BoNT/B intoxication [START_REF] Desplantes | Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor[END_REF]. Interestingly, pretreatment with PPMP decreased BoNT/B binding by 64 ± 3% (n= 3 independent experiments) (Fig. 1D). These results indicate that GT1b can still potentiate BoNT/B binding under limited membrane diffusion and suggest that there is a pool of SYT2 and GT1b co-receptors in sufficient proximity to allow toxin binding in the absence of lateral diffusion in the plane of the membrane.

Quantification of the effect of GT1b on BoNT/B binding to SYT2. We compared the affinity and the kinetic binding constants of the interaction between BoNT/B and SYT2 expressed in exosomes, in the presence and absence of GT1b, using SPR.

Insertion of exogenous GT1b in exosomal membranes induced a 80-fold increase in affinity (K D = 0.5 nM, Table 1 and(24)) compared to the condition in the absence of GT1b (40 nM). This increment is driven by a 20-fold increase in k on (1.5 ± 0.2 versus 31 ± 12 x10 4 M -1 .s -1 ) and a 4-fold decrease in k off (5.6 ± 0.1 versus 1.5 ± 0.6 x 10 -4 s -1 (Table 1). An estimation of the contribution of electrostatic interactions was carried out by increasing salt concentration in the running buffer. As shown in Table 1, moderate salt concentration increase (350 mM NaCl) induced a dramatic decrease in the association kinetic constant of BoNT/B with SYT2/GT1b, whereas it hardly influenced the k on on SYT2. Under these conditions, a similar on-rate (k on ) was observed for BoNT/B binding to both SYT2/GT1b (0.8 ± 0.2 x10 4 M -1 .s -1 ) and SYT2 (0.8 ± 0.1 x10 4 M -1 .s -1 ), thus losing the potentiation by GT1b. In contrast, the increment in ionic strength slightly increased the dissociation rate (k off ) of BoNT/B from both SYT2 (5.6 to 18.4 x10 -4 s -1 ) and SYT2GT1b (1.5 to 3.1 x10 -4 s -1 ) exosomes (Table 1). Taken together these results suggest that GT1b strongly increases BoNT/B affinity for SYT2, mainly via electrostatic interactions. Moreover, BoNT/B binding to SYT2 alone, as well as to SYT2/GT1b can be fitted using a simple homogenous 1:1 model (Fig. S1C, [START_REF] Desplantes | Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor[END_REF]), consistent with a single receptor site as if SYT and GT1b were closely positioned or already preassembled.

The juxta-membrane domains of SYT1 and 2 contain a conserved glycosphingolipid binding sequence and directly interact with GT1b. A careful examination of the complete N-terminal sequences of SYT revealed that SYT1 and SYT2 share a consensus glycosphingolipid binding motif [START_REF] Fantini | Prediction of glycolipid-binding domains from the amino acid sequence of lipid raft-associated proteins: application to HpaA, a protein involved in the adhesion of Helicobacter pylori to gastrointestinal cells[END_REF] [START_REF] Fantini | Molecular basis for the glycosphingolipid-binding specificity of alpha-synuclein: key role of tyrosine 39 in membrane insertion[END_REF] in their membrane proximal region, illustrated in grey in Fig. 2A. This typical sequence K/R/H(X1-4)-F/Y-(X4-5)-K/R/H is composed of a central hydrophobic amino acid flanked by basic amino acids or histidine at defined positions and mostly overlaps with the BoNT/B binding site (F 39 -L 50 for SYT1 and F 47 -I 58 for SYT2) that was formerly determined by structural analysis [START_REF] Chai | Structural basis of cell surface receptor recognition by botulinum neurotoxin B[END_REF]. To assess whether this motif is a functional glycosphingolipid-binding domain, we used the Langmuir monolayer method and investigated the binding, to various gangliosides, of synthetic SYT1 (pSYT1 [START_REF] Joughin | Action-at-a-distance interactions enhance protein binding affinity[END_REF][START_REF] Lesovoy | Specific membrane binding of neurotoxin II can facilitate its delivery to acetylcholine receptor[END_REF][START_REF] Nishiki | The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a[END_REF][START_REF] Gil | Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes[END_REF][START_REF] Herreros | Lipid microdomains are involved in neurospecific binding and internalisation of clostridial neurotoxins[END_REF][START_REF] Muraro | The Nterminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane[END_REF][START_REF] Vrljic | Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1[END_REF][START_REF] Champagne | Binding of GM1 ganglioside to a synthetic peptide derived from the lysosomal sphingolipid activator protein saposin B[END_REF][START_REF] Melkonian | Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated[END_REF][START_REF] Kabayama | Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance[END_REF][START_REF] Martinez | GM1 specifically interacts with alpha-synuclein and inhibits fibrillation[END_REF][START_REF] Mclaurin | Structural transitions associated with the interaction of Alzheimer beta-amyloid peptides with gangliosides[END_REF] and SYT2 (pSYT2 49-60) peptides (Fig. S2), corresponding respectively to the predicted juxtamembrane glycosphingolipid-binding domain of SYT1 and 2. Both wild type peptides, but not the scrambled sequences (pSYT scr) (Fig. S2), interacted with GT1b (Fig. 2B). The peptides bound GT1b better than other gangliosides (Fig. 2C and D) such as GM1, GM3 and did not bind the sphingolipid lyso-lactosylceramide.

The specificity of the binding was confirmed by SPR using single bilayer liposomes, immobilized on a hydrophobic sensor chip. For these experiments, longer SYT peptides (pSYT1 32-57, pSYT2 40-66), as well as peptides containing amino acid substitutions (pSYT1 32-57 K 43 A, F 46 A, K 52 A, pSYT2 40-66 F 54 A, F 55 A) that disrupt the consensus site, were synthesized (Fig. S2). pSYT1 and pSYT2, but not mutated peptides, interacted with GT1b in liposomes (Fig. 2E andF). Affinity constants from equilibrium analysis of peptide interactions with the GT1b-containing liposomes were determined, yielding K Ds of 3.1 ± 0.2 and 3.3 ± 0.7 µM for pSYT1 and pSYT2 respectively (n = 4 independent experiments). No binding occurred to phosphatidylserine or phosphatidylglycerol-based liposomes that have similar negative charges to GT1b-containing liposomes (Fig. 2E andF), indicating that recognition of GT1b by pSYT involves more than a simple electrostatic attraction via the three negative charges of GT1b sialic acid groups.

GT1b promotes the formation of an alpha-helix in SYT. We performed CD spectroscopy to determine whether GT1b-binding drives conformational changes in SYT. The SYT1 and SYT2 peptides (pSYT1 41-52 and pSYT2 49-60), encompassing the sphingolipid-binding domain displayed random structure in solution (Fig. 3A and B, dashed line). In the presence of GT1b, wild type peptides exhibited two characteristic minima at 208 and 222 nm typical of an α-helix, while no conformational change was noticeable with scrambled peptides (Fig. S3 A andB).

Addition of GT1b to the longer SYT2 peptide used in SPR also induced the formation of an alpha helix (Fig. S3C). We consequently determined the relative concentration of ganglioside required to induce structural transitions in SYT2. The αhelical transition appeared at a molar ratio of 1:0.5 (peptide:GT1b), reaching a maximum at a molar ratio of 1:3 (Fig. S3D). Altogether these results indicate that the extra-cellular juxtamembrane domain of SYT1 and 2 effectively binds GT1b and that binding induces a transition from a random to an α-helical structure.

Modeling of the SYT/GT1b interaction.

To further assess structural features of the SYT/GT1b complex, we docked GT1b with SYT1 41-71 and SYT2 45-80 amino acid sequences encompassing in continuum their juxta-and TM domains (Fig. 4). In water, these peptides did not fold into a well-defined structure and appeared mostly disordered. When mixed with a model membrane containing ceramide, the TM region of the peptide formed an -helix. When ceramide was replaced by GT1b, the extracellular part of the peptide adopted an -helical structure that was clearly induced by the carbohydrate part of GT1b (Fig. 4). At this stage, the complex displayed a highly complementary structure with a free binding energy of -167.3 and -168 kJ/mole for SYT1 and SYT2 respectively. About 2/3 of this energy (-115.3 and -114) kJ/mole for SYT1 and SYT2 respectively corresponds to extramembranous interactions (Fig. S4B), whereas the remaining third was due to a remarkable fit between the ceramide part of the ganglioside and the TM region of SYT peptides (Fig. 4). The sphingosine and fatty acid chains of the ceramide moiety of GT1b are highly twisted so that they wrap around the TM domain of SYT (Fig. 4). This configuration explains why the membrane-embedded region of the complex has such a high impact (-52/54 kJ/mole) on the energy of interaction of the complex. The complex is stabilized by a combination of van der Waals forces and hydrogen bonds between the polar parts of GT1b and SYT1/2, and by van der Waals forces linking the ceramide part of GT1b and the TM domain of SYT1/2 (Fig. S4, Table S2). In addition, W 58 and W 66 of SYT1 & 2 located at the bilayer interface strongly interacts with both the glycone and ceramide moieties of GT1b (Fig. S4A).

The minimal distances between SYT1/2 residues and GT1b, including hydrogen atoms, were in the 2-3 Å range (Table S2), consistent with the high energy of interaction of each complex (Fig. S4B).

Involvement of the ceramide domain of GT1b in the SYT/GT1b interaction.

Prompted by the modelling data we probed for a direct interaction between SYT and GT1b in a cis configuration (i.e. in the same membrane). For this purpose, the Cterminal part of the extracellular domain of SYT2 was fused to an acyl group to allow membrane insertion (peptidoliposomes, Fig. S5A). The acyl group was either a myristyl (Myr) or a farnesyl group (Far). The rational was that, a Myr group will mimic the TM domain of SYT allowing hydrophobic interaction with the ceramide of GT1b whereas the Far group will not, due to lateral methyl groups that should interfere with the docking to the ceramide part of GT1b [START_REF] Levental | Greasing their way: lipid modifications determine protein association with membrane rafts[END_REF]. Molecular modelling (Fig. S5C, left and middle) confirmed the similarity of the docking between GT1b and both TM-SYT2 and Myr-SYT2. In contrast and as expected, when the Myr was replaced by a Far moiety (Fig. S5C, right) the latter docked to the opposite face of the GT1b ceramide than Myr and the exposed ceramide face was different.

We used spectroscopy to monitor the intrinsic fluorescence of a single tryptophan (W 66 ) residue, located at the lipid-water interface and remarkably sensitive to the polarity of the environment. Soluble SYT2 peptide has maximum peak intensity at 351 nm (Fig. S5B dashed line). Upon insertion of Myr or Far pSYT2 in liposomes the maximum emission peak shifted to 339 nm, indicating that W 66 is partially buried in a hydrophobic environment at the interface of the lipid bilayer (Fig. S5B solid line, Fig.

5A and B black line), and that the nature of the hydrophobic tail of SYT2 has no influence on the positioning of SYT2 W 66 relative to the membrane.

Whereas addition of lysolactosylceramide to Myr-pSYT2 peptidoliposome had no effect on the spectra (Fig. 5 A green dotted line), GT1b induced a decrease in fluorescence (24 ± 4% n= 4 independent experiments, Fig. 5A red line). This quenching of tryptophan fluorescence induced by GT1b is compatible with the direct interaction scheme presented earlier and especially the molecular modelling data showing that the W 66 of Myr-pSYT2 interacts with the beta-linked glucose ceramide bond of GT1b and the sia 7 (Fig. S4A). The decrease in fluorescence intensity observed with GT1b could be due to GT1b binding inducing α-helix formation in SYT2. The helix would reduce the distance between W 66 and a charged amino acid such as K 60 , decreasing electron transfer and thus the emission energy of tryptophan [START_REF] Loura | Interaction of peptides with binary phospholipid membranes: application of fluorescence methodologies[END_REF][START_REF] Liu | Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins[END_REF].

Addition of GT1b to Far-pSYT2 peptidoliposomes did not modify the peak intensity (3.3 % of variation n= 3 independent experiments) but induced a blue shift from 339 ± 1.7 nm to 332 ± 1 nm (n=3 independent experiments), indicating a more hydrophobic environment and suggesting that the tryptophan residue penetrates into the lipid bilayer (Fig. 5B red trace). This result is consistent with molecular modelling in the presence of GT1b, where in Myr-pSYT2, the indol cycle of W 66 adopts a position parallel to the membrane plane, while in Far-pSYT2 the same indol cycle is perpendicular to the membrane plane and is positioned below the membrane surface (Fig. 5D). These results suggest that the interaction of the SYT membrane anchor with the ceramide part of GT1b is involved in the orientation of the extracellular domain of SYT2.

In the light of these findings, BoNT/B binding experiments were carried out by SPR using Myr and Far-pSYT2 incorporated in liposomes. In the absence of gangliosides, we observed similar BoNT/B binding kinetics to both types of peptidoliposomes, confirming that the accessibility of the SYT2 peptide to BoNT/B is not modified by the nature of the acyl anchor (Fig. 5C black traces). The addition of GT1b potentiated BoNT/B binding to both types of peptidoliposomes (Fig. 5C red traces) and confirmed that the reconstitution system used allows investigation of the GT1b potentiation of BoNT/B binding. Interestingly however, the association and dissociation kinetics of BoNT/B binding to Far-pSYT2 relative to Myr-pSYT2 liposomes were modified, notably with a significant 2.5-fold higher dissociation rate (k off = 7.4 x 10 -3 ± 1.4 s -1 and 2,9 x 10 -3 ± 0,6 s -1 ; results are mean ± SEM) from Far-pSYT2 relative to Myr-pSYT2 liposomes respectively. Altogether, these results indicate that cis interactions can occur between SYT2 peptide and GT1b in the same membrane plane and that intramembrane interactions between the ceramide group and the membrane anchor of SYT2 contribute to a precise positioning of the extracellular SYT2/GT1b complex favoring BoNT/B interaction.

Assembly of a SYT/GT1b complex plays a crucial role in BoNT/B binding

To pursue the necessity for preassembly of a SYT/GT1b complex to constitute the BoNT/B receptor, we sought to identify a mutation that perturbs SYT binding to GT1b, without inhibiting SYT binding to BoNT/B in the absence of GT1b. We focused on SYT1 which has a stronger GT1b requirement for BoNT/B binding. We therefore mutated K 52 in SYT1, a highly conserved amino acid predicted to interact with GT1b (Fig. 4B and Fig. S4A) but not with BoNT/B [START_REF] Chai | Structural basis of cell surface receptor recognition by botulinum neurotoxin B[END_REF][START_REF] Jin | Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity[END_REF][START_REF] Elliott | Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models[END_REF][START_REF] Berntsson | Structure of dual receptor binding to botulinum neurotoxin B[END_REF][START_REF] Tao | Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors[END_REF]. SPR analysis showed that the K 52 A mutation in pSYT1 32-57 drastically decreased by 63 ± 13 % (n = 6 independent experiments) (Fig. 6A) its interaction with GT1b, whereas a mutation in another lysine residue (K 43 ) has no significant effect. A control peptide corresponding to the juxtamembrane extracellular domain of SYT9 (pSYT9 27-53), that do not contain a consensus ganglioside-binding motif, accordingly, did not show any interaction.

In order to address the effect of K 52 A on BoNT/B binding in the absence of GT1b, we immobilized both wild type and mutant pSYT1 32-57 on SPR sensor chips (Fig. 6B).

BoNT/B interaction with wild type pSYT1 produced square signals with very fast k on and k off , characteristic of low affinity interactions. These signals were specific as binding of BoNT/E was not detectable (Fig. 6B). Although GT1b interaction was significantly inhibited by the K 52 A mutation, (Fig. 6A), surprisingly, BoNT/B binding to pSYT1 32-57 K 52 A in the absence of GT1b was enhanced (50 ± 3 %, n = 3 independent experiments) (Fig. 6B). This effect could be explained by the mutation annulling electrostatic repulsion between the positive dipole found in the C-terminus binding domain of BoNT/B (31) and the positive charge of K 52 .

We then evaluated the effects of the K 52 A mutation in the full-length protein by pull down experiments using recombinant GST-SYT1 in detergent. As expected, BoNT/B binding to wild type SYT1 was only observed in the presence of GT1b (Fig. 6C) [START_REF] Dong | Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells[END_REF], whereas the interaction was practically eliminated by the K 52 A mutation.

We then investigated by SPR the binding of BoNT/B to full-length wild type and mutated GST-SYT1 K 52 A reconstituted in proteoliposomes and immobilized on SPR sensorchips, in the presence of GT1b. Although BoNT/B binding to mutant pSYT1 32-57 K 52 A was significantly enhanced in the absence of GT1b (Fig. 6B), the K 52 A mutation induced a strong decrease in BoNT/B interaction with a membrane-inserted full-length protein in the presence of GT1b. No binding of BoNT/B was observed in the absence of GT1b (Fig. 6D, lower traces). These data clearly show that when preassembly of a SYT/GT1b complex is perturbed, subsequent BoNT/B binding is strongly reduced. They are consistent with the proposal that the SYT/GT1b complex constitutes the BoNT/B receptor.

To further investigate whether BoNT/B binding requires preassembly of a GT1b/SYT complex in a cellular context, we investigated the effects of the SYT1 K 52 A mutation in PC12 cells. PC12 cells were transfected with SYT1 or SYT1 K 52 A, loaded with GT1b and incubated with BoNT/B. The binding of BoNT/B was virtually abolished in cells expressing SYT1 K 52 A when compared to control cells expressing SYT1 WT (81.4 ± 4.7% of WT-SYT1 versus 4.0 ± 2.9% of SYT1 K 52 A-transfected cells were positive for BoNT/B labeling) (Fig. 7A, B andD). No BoNT/B binding (0.0 ± 0.0%) was observed on cells transfected with the same carrier plasmid devoid of SYT (Fig. 7A andD). In order to exclude the possibility of a potential mistargeting of SYT1 K 52 A that would hinder BoNT/B binding, we performed orthogonal projections of synaptotagmin labelling in both WT-SYT1 and SYT1 K 52 A-transfected cells. No apparent differences in the subcellular distribution of SYT between WT-SYT1 and SYT1 K 52 A-transfected cells was observed (Fig. 7C). These results support the conclusion that preassembly of a SYT/GT1b complex is required for BoNT/B to bind to the cell surface.

Discussion

BoNT/B displays remarkable specificity for peripheral nerve terminals conferred by two receptor molecules: the synaptic vesicle protein SYT (1 or 2) and the ganglioside GT1b, which although ubiquitous is particularly enriched in axonal terminals. The Cterminal domain of BoNT/B contains two distinct binding pockets for (1) an α-helical segment of SYT and (2) a ganglioside [START_REF] Berntsson | Structure of dual receptor binding to botulinum neurotoxin B[END_REF]. This is consistent with the currently predominant schema for independent interaction with dual receptors, in which BoNT/B initially interacts with GT1b, then diffuses in the membrane plane until it meets its protein receptor [START_REF] Montecucco | How do tetanus and botulinum toxins bind to neuronal membranes?[END_REF]. Binding to SYT would then ensure toxin internalization through receptor-mediated endocytosis [START_REF] Schiavo | Structural biology: dangerous liaisons on neurons[END_REF][START_REF] Pellett | Botulinum Neurotoxins Can Enter Cultured Neurons Independent of Synaptic Vesicle Recycling[END_REF]. In this study we first reinvestigated, in a membrane context, BoNT/B binding to each receptor separately and quantified the increase of affinity due to the presence of GT1b. Binding of BoNT/B to PC12 cells expressing or not SYT was addressed in the presence or absence of the glucosyl-ceramide synthase inhibitor PPMP in order to drastically decrease ganglioside level in these cells [START_REF] Dong | Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells[END_REF]. Careful analysis of binding to PC12 cells and membrane vesicles failed to detect an interaction of BoNT/B with GT1b alone. These findings corroborate previous studies on membrane vesicles or cultured cells not expressing SYT [START_REF] Dong | Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells[END_REF][START_REF] Dong | Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons[END_REF][START_REF] Kozaki | Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins[END_REF][START_REF] Sun | Receptor binding enables botulinum neurotoxin B to sense low pH for translocation channel assembly[END_REF]. Thus BoNT/B may bind to GT1b with very low affinity (>µM) and rapid dissociation kinetics [START_REF] Stern | A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency[END_REF], that rule out detection in assays requiring washing procedures. The absence of BoNT/B binding detection to membrane-associated GT1b may be due to the fact that it has a single gangliosidebinding pocket, in contrast to tetanus toxin, which has two and which displays nanomolar affinity for GT1b (37) [START_REF] Chen | Gangliosides as high affinity receptors for tetanus neurotoxin[END_REF].

In contrast we readily detected BoNT/B binding to SYT2 expressed in PC12 cells depleted of GT1b, whereas in similar conditions BoNT/B binding to PC12 cells expressing SYT1 cannot be detected in the absence of added gangliosides [START_REF] Dong | Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells[END_REF]. We then used exosomes expressing recombinant SYT2 to quantify the potentiation by GT1b of BoNT/B binding, in terms of K D and kinetic constants. This method has revealed an affinity for BoNT/B and association / dissociation rate constants, very close to those found in native membranes [START_REF] Desplantes | Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor[END_REF][START_REF] Dolly | Localization of sites for 125I-labelled botulinum neurotoxin at murine neuromuscular junction and its binding to rat brain synaptosomes[END_REF]. SPR measurements in exosomes expressing SYT2 indicated a K D = 40 nM in the absence of GT1b. The addition of exogenous GT1b led to an 80-fold increase in affinity. The large increase in the association rate conferred by GT1b was shown to be dependent on electrostatic interactions as it was totally abolished by an increase in ionic strength of the medium. Gangliosides in neuronal cell membranes contribute strongly to the negative surface charge as they contribute about 75% of plasma membrane sialic acids [START_REF] Schnaar | Sialic acids in the brain: gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration[END_REF]. Moreover, it has been shown that, long range electrostatic interactions between non-contacting residues of the binding partners can play a critical role in the formation of high affinity complexes [START_REF] Joughin | Action-at-a-distance interactions enhance protein binding affinity[END_REF] [START_REF] Lesovoy | Specific membrane binding of neurotoxin II can facilitate its delivery to acetylcholine receptor[END_REF]. Thus as suggested earlier, gangliosides in anionic microdomains could have a major role in increasing BoNT/B affinity for neuronal membranes through electrostatic interactions involving a positive pole found in the BoNT/B C-terminal domain [START_REF] Fogolari | Electric dipole reorientation in the interaction of botulinum neurotoxins with neuronal membranes[END_REF].

BoNT/B has been estimated to have at least 100-fold higher affinity for SYT2 versus SYT1 (17) [START_REF] Tao | Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors[END_REF], whereas the addition of GT1b reduces this difference 10-fold [START_REF] Nishiki | The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a[END_REF].

Since GT1b enhances BoNT/B affinity for SYT2 by a factor of 80, by extrapolation GT1b would increase the affinity of BoNT/B for SYT1 by a factor of 800. However, it was not possible to quantify the increase in BoNT/B affinity conferred by GT1b to SYT1 as binding of the toxin to SYT1 in a membrane context and in the absence of gangliosides is not detectable [START_REF] Dong | Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells[END_REF]. The fact that the potentiation by GT1b is more pronounced for SYT1 is particularly important, as SYT1 appears to be the principal BoNT/B receptor in humans [START_REF] Peng | Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins[END_REF].

The fact that we were unable to detect BoNT/B binding to GT1b alone, and that GT1b appears to act principally by potentiating binding to SYT, along with SPR data showing that BoNT/B interaction with SYT and GT1b can be fitted with a 1:1 ligand : receptor model, led us to consider the possibility that BoNT/B binds simultaneously, rather than sequentially, to both GT1b and SYT. GT1b-dependent binding to SYT occurred in conditions that severely restrict lateral diffusion, suggesting that a pool of SYT is in sufficient proximity to GT1b to allow BoNT/B to simultaneously bind to both co-receptors. This conclusion is in agreement with independent observations showing that BoNT/B but also BoNT/A are found associated with their corresponding receptors in glycolipid-rich microdomains (44) (45) [START_REF] Muraro | The Nterminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane[END_REF].

We then identified a consensus site for ganglioside binding located in extracellular juxtamembrane domains of SYT 1 and 2 and confirmed that peptides covering this region interact directly with GT1b.The site did not display specificity for any particular ganglioside, recognizing other major species in the nerve terminal such as GM1, although the strongest interaction was with GT1b. Interestingly, full length SYT1 expressed in insect cells has been reported to bind GM1 and GD3 in lipid overlay assays, although the binding domain was not identified [START_REF] Vrljic | Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1[END_REF]. We did not detect any interaction with acidic phospholipids, ruling out a simple electrostatic interaction between ganglioside sialic acid and basic residues of the used peptides. The affinity of SYT peptides for the oligosaccharide moiety of GT1b was about K D = 3 µM, comparable with that of saposin for GM1 [START_REF] Champagne | Binding of GM1 ganglioside to a synthetic peptide derived from the lysosomal sphingolipid activator protein saposin B[END_REF]. However, binding parameters measured in a trans configuration using a soluble peptide do not take into account additional hydrophobic interactions within the membrane phase.

CD analysis indicated that whereas peptides were unstructured in solution, GT1b induced an increase in α-helical content in both SYT1 and 2. This data was consistent with modeling which showed considerable structural complementarity between GT1b and the juxtamembrane region of SYT1 or 2. The ganglioside can wrap itself round the TM of SYT, and interact with the extracellular juxta-membrane domain of SYT that forms an α-helix.

The potentiating effect of GT1b on BoNT/B binding to SYT has only been observed when the two receptor components were in the same membrane plane, be it a micelle, liposome or native membrane (32) [START_REF] Kozaki | Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins[END_REF]. Experiments monitoring the fluorescence of an intrinsic tryptophan residue located at the membrane solvent interface confirmed a cis interaction between GT1b and SYT2 in a membrane context. Our data indicated that the nature of the acyl group anchoring SYT in the membrane, modified the environment of the tryptophan when GT1b was present, consistent with hydrophobic interactions between the peptide membrane anchor and the ceramide moiety. Furthermore, substitution of myristyl by farnesyl, precluding appropriate intramembrane interactions with ceramide (49) [START_REF] Fantini | Prediction of glycolipid-binding domains from the amino acid sequence of lipid raft-associated proteins: application to HpaA, a protein involved in the adhesion of Helicobacter pylori to gastrointestinal cells[END_REF], influenced BoNT/B binding, notably by accelerating dissociation kinetics. These data, associated with modeling, suggest that interactions between the membrane anchors of GT1b and SYT contribute to assembly of the high affinity BoNT/B receptor site. The association of SYT TM and ceramide confirms initial studies suggesting a SYT/GT1b interaction through hydrophobic domains [START_REF] Kozaki | Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins[END_REF].

The involvement of the hydrophobic domains of SYT and GT1b regarding BoNT/B binding is highlighted by the following observations: (1) SYT1 binds BoNT/B in presence of GT1b only when the TM domain is present and (2) the sugar domain of the ganglioside is not sufficient to potentiate BoNT/B binding to SYT1 or SYT2 [START_REF] Dong | Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells[END_REF] (17) [START_REF] Berntsson | Structure of dual receptor binding to botulinum neurotoxin B[END_REF]. Altogether these results suggest that the interaction of SYT and GT1b through intramembrane and extracellular domains are required for BoNT/B binding.

Next we addressed the importance of preassembly of a GT1b / SYT1 complex for BoNT/B binding. The K 52 residue is located outside of the reported SYT binding pocket of BoNT/B [START_REF] Elliott | Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models[END_REF], but within the consensus sequence for ganglioside binding and interacts with GT1b in modeling studies. In fact, we demonstrated that the K 52 A mutation block GT1b potentiation of BoNT/B binding to full length SYT1, in vitro or expressed at the surface of intact PC12 cells. This parallels insulin receptors, which bind GM3 ganglioside upon export to the plasma membrane. In this case the first extracellular lysine following the TM domain is also crucial for ganglioside binding [START_REF] Kabayama | Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance[END_REF].

Structural data shows that the short amphipathic helical region, adjacent to the TM domain of SYT1 and SYT2, interacts with BoNT/B in a similar manner [START_REF] Elliott | Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models[END_REF]. The strong dependency on GT1b for BoNT/B binding to SYT1 is thought to be due to the nature of the amino acids (L 50 and H 51 ) located next to K 52 , as substitution by the corresponding amino acids of SYT2 rescued BoNT/B binding in the absence of GT1b [START_REF] Chai | Structural basis of cell surface receptor recognition by botulinum neurotoxin B[END_REF]. Our data, showing that K 52 plays a crucial role in GT1b binding to SYT1, suggest that the interaction of SYT1 with GT1b reduces the inhibitory effects of these vicinal amino acids and induces the formation of an α helix. Interestingly, a similar phenomenon of alpha helix structure induced by the head group of gangliosides, has been reported for α-synuclein [START_REF] Martinez | GM1 specifically interacts with alpha-synuclein and inhibits fibrillation[END_REF] and Alzheimer beta amyloid peptide (52) [START_REF] Matsuzaki | How do membranes initiate Alzheimer's Disease? Formation of toxic amyloid fibrils by the amyloid beta-protein on ganglioside clusters[END_REF]. Our data suggest that ganglioside could also modulate the secondary structure of a transmembrane protein. In this case, the amphipathic helix would provide structural complementarity between the SYT1 juxtamembrane domain and the SYT binding pocket of BoNT/B, involving mostly hydrophobic residues and complementary salt bridges [START_REF] Berntsson | Structure of dual receptor binding to botulinum neurotoxin B[END_REF] [START_REF] Elliott | Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models[END_REF].

We thus propose that upon exocytosis of SYT, the SYT/GT1b complex would rapidly assemble, given the large molar ganglioside: SYT ratio at the plasma membrane.

BoNT/B would then bind to the N-terminal part of SYT exposed to the extracellular medium and bound to GT1b (Fig. 8). Alternatively BoNT/B could bind to a resident plasma membrane pool of SYT (54) (55) [START_REF] Willig | STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis[END_REF] pre-assembled with GT1b.

Interestingly, in view of the fact that SYT can also bind GM1, a similar model could be applied to BoNT/DC, a botulinum subtype toxin that uses GM1 as a co-receptor with SYT [START_REF] Zhang | Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC[END_REF]. The simultaneous binding of BoNT/B to a pre-assembled co-receptor could explain thus the high affinity and specific binding to the external surface of the presynaptic membrane.

The current results suggest that GT1b may play a triple role in BoNT/B binding: i) as an electrostatic attractor, ii) in the SYT / GT1b receptor complex, forming and presenting an α-helical segment to the SYT binding pocket and iii) in stabilizing the toxin receptor complex via the ganglioside binding pocket. Further studies will be necessary to ascertain what happens when the toxin meets the assembled SYT / GT1b complex as certain amino acids crucial for SYT binding to BoNT/B are also involved in the SYT / GT1b interaction. Complementary studies are required to determine whether the toxin could displace the GT1b bound to SYT and if so whether the hydrophobic loop of BoNT/B positioned between the SYT and the GT1b binding pockets is involved.

Experimental procedures

Reagents. All used reagents are listed in SI Experimental procedures.

Cloning. Rat SYT 1 and 2 coding sequences were amplified by PCR from a commercial Y2H adult rat brain cDNA library (Origine) and inserted in the bicistronic vector encoding PIRES-EGFP (Clontech). SYT 2 was inserted in non-modified PIRES-EGFP and a myc tag was included in the reverse primer to generate a Cterminally tagged SYT2. SYT1 was inserted between EcoR1 and Sal1 sites in modified PIRES2-EGFP in which a myc tag was inserted at the beginning of the multiple cloning site between Xho1 and EcoR1. Directed mutagenesis was then used to introduce the K 52 A mutation in SYT1 and 2 respectively. For GST constructs, SYT1 coding sequences were inserted between EcoR1 and Sal1 sites of pGEX4T (GE-Healthcare).

Recombinant protein expression. GST-SYT1 was expressed in BL21 E. coli following classical procedures and purified on Glutathione-Sepharose in 20 mM

Tris/HCl pH 7.4, 140 mM NaCl, 0.1% Triton X-100 buffer.

Exosomes. Exosomes expressing SYT2 and AMA1 were produced and purified as described earlier [START_REF] Desplantes | Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor[END_REF] and kept frozen before use.

Liposomal preparations. Liposomes for SPR lipid-binding specificity experiments:

Liposomes were prepared using mixtures of DMPC with 4% GT1b, 12% PS or 12% PG or 100% of DMPC for control liposomes (850 nmoles). Solvent was evaporated and lipids rehydrated in TBS (25 mM Tris/HCl pH 7.4, 150 mM NaCl). Liposomes were prepared by extrusion at 55°C through 100-nm polycarbonate filters using a basic Lipofast apparatus (Avestin, Ottawa). For peptidoliposomes and proteoliposomes see SI Experimental Procedures.

Langmuir balance. SYT peptide binding to gangliosides was determined using the Langmuir-film balance technique essentially as previously described using a fully automated microtensiometer (μTrough SX; Kibron Inc.) [START_REF] Scala | Interaction of Alzheimer's beta-amyloid peptides with cholesterol: mechanistic insights into amyloid pore formation[END_REF]. Briefly, interactions were monitored at 20 ± 1°C by surface pressure modification of monomolecular films of gangliosides re-suspended in chloroform/methanol (1:1 vol/vol) and spread on pure water sub-phases (800 μl). The initial surface pressure of the monolayers was set between 12 and 15 mN/m and the accuracy of measurement conditions was ± 0.25 mN/m. SYT peptides were injected (final concentration of 10 μM) into the aqueous phase underneath the ganglioside monolayer until equilibrium was reached and kinetics of surface pressure changes were followed by real-time measurements.

Data were analyzed with the FilmWareX program (version 3.57; Kibron Inc.). Circular dichroism. CD spectra were obtained at 20 °C in 1 mm path length quartz cuvettes using a JASCO model J-810 spectropolarimeter. 30 µM SYT peptide solutions were prepared in filtered 10 mM phosphate buffer pH 7.6. Gangliosides were added at a final concentration of 90 µM unless otherwise indicated and samples were incubated 10 min before CD analysis.

CD spectra were obtained between 190 nm and 260 nm at 1 nm intervals. Each CD spectrum shown is the average of three scans with a scanning speed of 20 nm / min. The spectra were corrected for contributions from buffer and GT1b as appropriate.

Data analysis was carried out using the online Dichroweb platform (59, 60) combined with the CDSSTR algorithm. For all CD spectra, the NRMSD parameter (a normalized standard deviation for each curve fit) was below 0.1.

Fluorescence Spectroscopy. Fluorescence spectra were recorded on a Cary

Eclipse fluorescence spectrofluorimeter (Varian, USA) with emission and excitation slit widths of 5 nm. Peptidoliposomes containing Myr-SYT2 or Far-SYT2 were diluted in TBS in a total volume of 0.5 ml and the intrinsic fluorescence of SYT2 was recorded in the absence or presence of GT1b or lyso-lactosyl ceramide (23 µM) that were added exogenously with a ganglioside / peptide ratio (mole/mole) of 3.

Excitation wavelength was 278 nm. Emission spectra were recorded from 300 to 400 nm, averaging 3 scans. All the spectra were corrected for the fluorescence of the corresponding blank (liposomes ± GT1b).

Immunofluorescence staining. PC12 cells deficient in SYT1 and 2 (PC12ΔSYT1), were cultured on poly-L-Lysine (10 μg/ml) treated coverslips (300,000 cells per well) in DMEM containing 5% FBS, 5% HS and 1% penicillin/streptomycin mixture. Where stated, 7.5 μM of the "glucosyl-ceramide synthase" inhibitor PPMP (DL-threo-1phenyl-2-palmitoylamino-3-morpholino-1-propanol) was added to the culture medium for 48h. Cells were transfected with the corresponding plasmids (pIRES-EGFP; pIRES-EGFP-SYT1 or pIRES-EGFP-SYT2) using Lipofectamine 2000 and following the manufacturers procedure. Forty hours after transfection and when appropriate, GT1b (10 µg/ml) was added to the wells in DMEM and incubated for 1.5 h at 37°C.

BoNT/B at the indicated concentrations or a mixture of BoNT/B and anti-SYT2 antibody (8G2b 1 µg/ml) were then added to the culture medium and incubated for 45 min at 37°C. After a first wash with the culture medium, additional washes were performed with PBS and cells were fixed in the dark at 4°C in 4% paraformaldehyde/PBS followed by NH 4 Cl washing steps. Non-specific binding was blocked with 0.2% (w/v) gelatine or 5% (v/v) goat serum in a PBS buffer containing 0.1% saponin. Anti-BoNT/B (0.5 µg/µl), anti-GFP (2 µg/ml), 1D12 anti-SYT (1 µg/ml) or anti-GT1b (1 : 1000) antibodies were then added for 45 min at 22°C. After subsequent washing, staining was visualized using secondary anti-rabbit Alexa 594 or 555 and anti-mouse Alexa 647 nm or 488 nm antibodies. Nuclei were detected using DAPI. analysed by immunoblot using anti-BoNT/B antibody at 0.5 µg/ml (Metabiologics).

Confocal microscopy and quantitative

Red Ponceau detection was used to ensure that equal amounts of bait protein were captured on the beads.

Fig. 1 .

 1 Fig. 1. BoNT/B binding to PC12 cells transfected with SYT2. (A)

Fig. 2 .

 2 Fig. 2. SYT interaction with gangliosides. (A) Glycosphingolipid binding motif in the

Fig. 3 .

 3 Fig. 3. GT1b induces conformational changes in SYT peptides in solution. Far-

Fig. 4 .

 4 Fig. 4. Molecular modelling of GT1b complexed with SYT1 and SYT2. (A and B)

Fig. 5 .

 5 Fig. 5. Involvement of the ceramide domain of GT1b in SYT2 / GT1b interaction. (A

Fig. 6 .

 6 Fig. 6. The juxtamembrane SYT1 lysine residue (K 52 ) is crucial for interaction with

Fig. 7 . 1 (

 71 Fig. 7. Mutation of the SYT1 juxtamembrane lysine (K 52 A) inhibits GT1b-dependent

Fig. 8 .

 8 Fig. 8. Schematic model for BoNT/B binding to neuronal membranes. (1) Upon

  /rsb.info.nih.gov/ij/). PPMP effects were presented as ratios of relative intensities to mean fluorescence values obtained in the absence of PPMP. Results are shown as mean ± SD and statistical analysis were done using Mann-Whitney U test. Results of BoNT/B binding to non-transfected, wild-type SYT1 and mutant SYT1-K 52 A expressing PC12 were presented as means ± SEM and « one-way » ANOVA followed by Bonferroni's post-hoc test was used for comparisons.

	descent gradients or Polak-Ribière conjugate gradients until convergence to machine
	precision. GT1b was then merged with energy minimized SYT1 or SYT2 peptides
	generated de novo with the amino acid database of Hyperchem. Initial conditions of
	analyses. Confocal images were acquired microscope and analysed using ImageJ well plates pre-treated with poly-L-lysine (10 μg/ml). Cells were transfected with on a Zeiss LSM780 SYT2 or carrier vector and treated when appropriate with PPMP as for immunofluorescence experiments. BoNT/B (5 nM) or a mixture of BoNT/B and the neutralizing anti-BoNT/B antibody (100 nM) were then added to the culture medium and incubated for 1h on ice. Cells were washed with PBS and fixed at 4°C in 4% paraformaldehyde/PBS. A 45 min consecutive blocking step with 0.056% saponin, 0,2% gelatine in PBS was preceded by 2 x 5 minutes NH 4 Cl washing steps. Anti-BoNT/B (0.5 µg/µl) was then added for 1 hour at 22°C in the blocking buffer. Three washing steps were performed with the blocking buffer before adding the secondary HRP-coupled anti-rabbit antibody for an additional hour at 22°C. Cells were washed 5 times with the blocking buffer and the HRP substrate TMB was added. The colorimetric reaction was stopped by H 2 SO 4 and the OD measured at 450 nm. Molecular modelling. In silico analyses were performed using Hyperchem and Molegro Molecular viewer as described (61). The initial coordinates of GT1b were gui.org/input/glycolipid (62), which uses the internal coordinate information of common glycosidic torsion angle values, orients the ganglioside perpendicular to the SYT 1 / 2 and GT1b (Sphingosine / C18:0) corresponded to minimized structures obtained with the Polak-Ribière algorithm. Docked SYT-GT1b complexes were subsequently submitted to iterative cycles of molecular dynamics using the CHARMM36 force field optimized for sphingolipids (63) and carbohydrates (64). Interaction energies were calculated from stable complexes after several 10 ns cycles using the « Ligand Energy Inspector » function of Molegro. Surface plasmon resonance spectroscopy (SPR). SPR measurements were conducted at 25°C at a flow rate of 30 µl/min on a Biacore 3000 or Biacore T200 (GE Healthcare Life sciences, Velizy-Villacoubray, France). For details see SI Experimental procedures Pull-down experiments. GST and GST-SYT1 (10 µg) were immobilized on glutathione Sepharose 4B resin. Washed beads were incubated in TBST (25 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with BoNT/B (10 nM) for 1 h at 4°C, in the presence or absence of GT1b (25 µg/ml). The beads were washed, and (http:/Cellular ELISA. PC12 cells (300.000 per well) were cultured directly on plastic 24 obtained from CHARMM-GUI Glycolipid Modeler (http://www.charmm-bound proteins boiled 5 min in SDS-sample buffer containing 10 mM DTT and
	membrane, and performs Langevin dynamics with a cylindrical restraint potential to
	keep the whole GT1b molecule cylindrical. In the next step, we included GT1b in a
	periodic box (30 x 30 x 50 Å) solvated with 1487 water molecules. The system was
	energy minimized 6 times switching alternatively between runs using steepest

Table 1

 1 Affinity of BoNT/B for SYT2 and SYT2 /GT1b exosomes Ds were determined with a 1:1 Langmuir model, as described in Materials and Methods using buffer containing physiological (150 mM) or higher NaCl (350 mM) concentrations. n represents independent experiments in 150 mM NaCl conditions and multiple determinations from 2 independent experiments in 350 mM NaCl condition. Values are means ± SEM.

		R e c e p t o r	NaCl (mM)	k on (x10 4 M -1 .s -1 )	k off (x10 -4 s -1 )	K D (x10 -9 M)	n
	BoNT/B	SYT2 SYT2 + GT1b	150 350 150 350	1.5 ± 0.2 0.8 ± 0.1 31.0 ± 12 0.8 ± 0.2	5.6 ± 0.1 18.4 ± 3.0 1.5 ± 0.6 3.1 ± 0.9	40.3 ± 0.5 228.0 ±31.9 0.5 ± 0.05 52.5 ± 19.6	6 4 3 6
	K

Acknowledgements. We thank Nouara Yahi and Raymond Miquelis for helpful discussion, Robert Mamoun (Ciloa SAS) for exosomes production. This work was financially supported by INSERM and Aix-Marseille University. PhD fellowships of A.

F. and R. D. were co-financed by INSERM and the Region SUD (PACA). Ciloa SAS co-funded the PhD fellowship of R.D. We thank the ANR (Agence Nationale de la Recherche) for financially supporting J.R.F.


Edoardo Marchini

Mohamed Darari

Luca Lazzarin

Rita Boaretto

Roberto Argazzi

Carlo Alberto Bignozzi

Philippe C Gros

Stefano Caramori

Recombination and regeneration dynamics in FeNHC( ii )-sensitized solar cells

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

Recombination and regeneration dynamics in Fe-NHC-sensitized DSSCs revealed incomplete injection and the detrimental role of photoinjected electrons recapture by the I 3

-form of the redox electrolyte on performance. Importantly, the use of additives in the electrolyte allowed to reach the best efficiency ever recorded for an iron-based DSSC.

Dye-sensitized solar cells (DSSCs) are popular photovoltaic devices because of their low cost and transparency . 1 Their principle is to harvest solar photons using a sensitizer, linked to a photoanode (coated with n-type TiO 2 semiconductor (SC)). Upon light excitation, the sensitizer promptly injects its electron into the SC. A redox mediator then regenerates the sensitizer's ground state and is in turn regenerated at the cathode thus completing the electric circuit. Ruthenium polypyridine complexes have long been considered as top sensitizers due to their ideal photophysical and redox properties , 2 reaching up to 11-14% conversion efficiency.

3 Despite all these impressive properties, ruthenium remains a scarce and expensive metal limiting the real-world industrial development. This has motivated the quest for alternative metals such as iron 4, 5 . Unfortunately, conventional Fe(II) polypyridyl complexes are characterized by an ultrafast (ca. 100 fs) deactivation of the MLCT manifold to metal-centered (MC) states. [6][7][8][9] Thus, the too short lived the excited state impedes injection into the SC conduction band. One of the most successful strategies employed to delay the deactivation is to increase the ligand field strength. [10][11][12][13][14][15][16] Significant advances have been recently reported by the use of Pyridyl-Nheterocyclic carbenes (NHC), 10, 13-16 resulting in notable improvement of 3 MLCT lifetimes up to 16 ps 13 for complex C1

(inset Figure 1) combining -donating NHCs and -accepting carboxylic groups to be covalently grafted to the TiO 2 surface. The first photovoltaic characterization of a Fe(II)-NHC complex, reported by some of us, 13, 17 revealed a weak power conversion efficiency (0.13%). In consideration of the excited state lifetime of C1 (16 ps), we expect a sufficiently efficient injection into the SC, reported to occur ideally within the fs time scale. 18 Thus it was of crucial importance to identify the bottlenecks that still limit the efficiency of Fe(II) sensitized solar cells. Herein, we report the unprecedented investigation of recombination and regeneration dynamics involving C1 (Inset Figure 1) adsorbed on either n-type TiO 2 or SnO 2 in the presence of I -/I 3 as electrolyte as well as a new electrolyte allowing to reach ca. 1% PCE that is the best efficiency ever reported for an iron-sensitized DSSC. Despite the presence of a single binding COOH group, FeNHC adsorbs strongly on TiO 2 leading to a well-defined MLCT absorption peak centred at 500 nm resulting in a nearly quantitative light harvesting at such wavelength (Figure S1). Similar harvesting efficiency is found with SnO 2 photoanodes (Figure S2). Since, as expected, C1 is not emitting in fluid solution nor when loaded on solid thin films, its spectroscopic energy (E 0-0 =2.26 eV) was evaluated from the onset of the MLCT band at 550 nm. The cyclic voltammetry (Figures S3-S4) carried out on the sensitized thin films, showed a quasireversible Fe(II)/(III) oxidation process (E 1/2 = 0.67 V vs SCE , ΔE peak ≈ 100 mV) . Thus the first excited oxidation potential E* ox was found at -1.6 V vs SCE ensuring ample driving force (ca. -0.9 eV) for injection into TiO 2 whose flat band potential is typically reported 19 to lie at ca. -0.7 V vs SCE in organic electrolytes containing lithium salts. A screening of the effect of TiO 2 morphology was performed in order to select the optimal substrate for cell efficiency. Commercial transparent TiO 2 (Greatcell Solar 18NR-T) composed of ca. 20 nm nanoparticles (Figure S5) was used with (BUL) and without (no BUL) a blocking underlayer produced by spin coating a 0.3 M Ti(OiPr) 4 on top of FTO (Fluorine-Tin-Oxide, Figure S6). Similar configuration was used in the case of semi-opaque paste (Greatcell Solar 18NR-AO) where ca. 20 nm particles tasked with dye adsorption are mixed with large light scattering particles in the 100 nm range (Figure S7). SnO 2 electrodes were fabricated in a similar fashion, by exploiting a colloidal paste and a blocking underlayer made by spin coating SnCl 2 . Different electrolytes were tested as well. The base formulation consisted in 0. MgI 2 to which different additives including GuNCS (Guanidinium thiocyanate) (el2) (Table S1) and 4-tBuPy (el3) were added. A commercial high stability electrolyte (Dyesol HSE) was tested as well.

Current density vs Voltage (J-V) curves in Figure 1 show that a substantial improvement in photovoltaic performances (from 0.49 to 0.75 % efficiency (η)) with the use of the blocking underlayer (BUL electrodes), consistent with the increased shunt resistance which implies lower dark current flowing between -0.1 and -0.3 V. The use of el2, containing GuNCS and 0.1 M MgI 2 further enhances performances, with Jsc=3.3 mA/cm 2 , Voc = 0.44 V, FF=0.63 and η≈1% (Table S1). This improvement was due to the presence of GuNCS which slightly increases fill factor and Voc, ostensibly helping to block recombination, while Mg 2+ , being a cation with a high charge density may adsorb on the TiO 2 surface and induce a surface excess of I -, speeding up dye regeneration. To the best of our knowledge this the best efficiency ever achieved in an iron sensitized DSSC. The use of opaque titania produces slightly better photocurrents, but marginally lower Voc and a similar efficiency was observed. The better photocurrent generated by the opaque paste is consistent with broader IPCE (Incident Photon to Current Conversion Efficiency) (Figure 2) thanks to the improved light scattering properties of 18NR-AO, but the conversion maximum around 50% was substantially identical to that observed with the transparent electrodes which were used for the spectroscopic investigation (vide infra). Thus the kinetic information extracted from transient spectroscopy can be safely extended to the case of opaque substrates. Under comparable conditions (BUL-el1), a transparent N 719 sensitized reference cell produced a broader and higher IPCE spectrum (max. IPCE ≈80 %), Jsc ≈13 mA/cm 2 and η = 3.1%

(Figures S8 and Table S1). The relatively small efficiency of the N 719 cell is due to lack of basic additives which enhance Voc and FF. This choice was motivated by the need of comparing C1 and N 719 under identical electrolyte conditions. It was indeed reported by Housecroft et al. 20 that the presence of basic additives greatly depresses photocurrent in Fe(II) sensitized solar cells. We indeed confirmed the same trend here (Figure S9). J-Vs and IPCE with el1 were also collected with SnO 2 based DSSCs where, despite the ca. 300 meV larger driving force for charge injection, we obtained a limiting photocurrent value ca. half that observed with BUL-el1, corresponding to an IPCE of ca. 25% and APCE (Absorbed Photon to Current Conversion Efficiency)≈30% (Figures S10). This suggested no thermodynamic limitation for charge injection into TiO 2 with respect to SnO 2 and the existence of fast recombination channels from SnO 2 , 21 where the blocking underlayer had basically no effect on the resulting J-V characteristics (Figure S11). ‡ ns/ms time scale. TAS spectra at early delays after the laser pulse are shown in Figure 3. The photogeneration of the oxidized form of C1 is practically instantaneous for our instrumental response (FWHM 7 ns), however a careful look at the TAS spectra at early delays evidences a spectral evolution that is substantially convolved with the laser pulse, resulting in the narrowing and the in minor blue shift of the MLCT bleaching associated to the disappearance of a weak absorption band rising for λ > 650 nm. Such dynamics is mostly evident by plotting the spectra during the first 4 ns after the laser pulse maximum (Figure S12), during which the FWHM of the bleaching band narrows from 91 to 73 nm while a blue shift of a few nm in the bleach minimum occurs. This spectral evolution could be assigned to the relaxation of a residual population of the lowest triplet state (T1). T1 could decay to the ground state either via non radiative pathways or by electron injection into TiO 2 . The lower than unity Φ inj extracted from APCE probably corroborates the first hypothesis. Nevertheless after ca. 40 ns such dynamics is complete, and no further change in the spectral shape is observed. This sets a lower limit for the injection rate constant of ca. 10 8 s -1

. The Fe(III) form of C1 created upon laser excitation is thus characterized by the bleached MLCT band that mirrors the ground state absorption band and by a weak and featureless absorption extending to the red, probably originated by the absorption of LMCT states and of electrons photoinjected into TiO 2 . In the absence of electron donor, the recovery of Fe(III) is power dependent and multiexponential, (Figure S13) stretching the time interval from a few µs to 0.1 s, owing to a distribution of electronic states in the semiconductor from which recombination occurs with different rates. At low laser energy (300 µJ/cm 2 /pulse) the decay could be fitted with two components having approximately equal amplitudes, with time constants on the order of 2×10 -3 s and 2 ×10 -2 s respectively. At higher excitation fluences, a third component with a time constant in the scale of 10 -4 s has to be added to account for the faster recombination as the number of electron/hole (Fe(III)) pairs in each TiO 2 nanoparticle increases. Nevertheless, even in the presence of the stronger excitation used in this work (ca. 18 mJ/cm 2 /pulse), the decay of the charge separated state is less than 80% complete on a time scale of 0.1 s. By contrast, when iodide is present in overall 0.8 M concentration, 75 % of Fe(III) is regenerated on a time scale of 10 µs. A residual amplitude (ca 4 mΔOD) of the 500 nm bleaching however does not recover on this and on longer time scales. The TAS spectra in the presence of el2 (omitting I 2 ) in the µs-ms range are shown in Figure 4. The amplification of residual small ΔOD for t> 10µs evidenced in Figure 3 requires the application of 10 kOhm input impedance which increases the signal gain of our spectrometer by a factor of 200. The dynamics in the first 300 µs is thus substantially convolved with the laser pulse. Within this time window, we observe the fast recovery of the bleaching, accompanied by a red shift of ca. 40 nm. In the blue region, an absorption band rises, with a local maximum around 490 nm followed by a steep increase for λ< 450 nm. In the red, starting from 600 nm a flat absorption originated by photoinjected electrons increases (herein we have to assume that no LMCT states of Fe(III) are contributing, consistent with fast regeneration of Fe(II) observed at earlier delays). In the hundreds of µs to s range, such spectral features are then maintained with a slight reduction in their respective amplitudes due to charge recombination. We explain the initially fast bleach recovery as due to Fe(III) reduction by iodide, which results in the formation of triiodide, responsible for the absorption in the blue, particularly for the steep band rising for λ<420 nm. 22 Upon dye reduction, electrons are free to accumulate in the conduction band, where in the absence of oxidized form of the mediator (except the one which is locally photogenerated) they persist for several ms (Figure S21). The persistence and the shift of the bleach is not compatible with the fast electron donation evident in Figure 3, and is thus assigned to Stark effect, 22 resulting from electron accumulation in the mesoscopic semiconductor. The observed spectral changes are indeed consistent with spectro-electrochemical measurements (Figure S18) showing, upon negative polarization of the photoanode in the dark, a blue shift of the ground state absorption , which translates, in the difference spectra, in a relatively narrow bleach peaking around 530 nm accompanied by an absorption band peaking around 480 nm. This absorption feature agrees with the absorption band observed in Figure 4 within the same wavelength range. TAS in the presence of the complete el2 (Figure S15) was partly disturbed by the strong absorption in the blue arising from the presence of I 3 -. It was nevertheless possible to confirm the presence of the Stark bleach, followed by the absorption of photoinjected electrons extending from 600 nm to 800 nm and beyond. The analysis of the recovery kinetics in such interval, compared to the previous case, where no I 3 -was initially present, allows to gain information about the dynamics of electron recapture by the oxidized mediator. In the absence of I 3 -the 750 nm absorption (Figure S21) is practically constant on a time scale of 20 ms, with only 6% of the initial ΔA amplitude recovering on such time scale. The small variation of the signal prevents the possibility of a reliable fitting. By contrast when 0. Please do not adjust margins Please do not adjust margins was added to the electrolyte a decay with a time constant of 5.6 ms was observed (Figure S22), setting the apparent electron recapture rate constant by the redox couple in the order of 2 ×10 2 s -1

. For comparison the N 719 electrode in the presence of the same el2 electrolyte showed a time constant of ca. 18 ms (Figure S23), indicating a substantially slower electron recapture by the oxidized redox relay. This difference could be due to the different surface potential imparted to TiO 2 by adsorption of a positively charged dye in the case of C1 (where the dissociation of one of the two carboxylic group may eventually reduce the complex charge from +2 to +1) (Figures S24-S27) and of a negative dye in the case of N 719. Cations present at the surface may eventually attract a local excess of iodide, instrumental to efficient regeneration, but also a surface excess of I 3 -which contributes to redox mediated recombination. The non-optimal shielding of the photoanode against recombination mediated by I 3 -may explain the strong effect of BUL in boosting cell performance in the case of C1.

The comparison with transient data obtained with N719 also shows that at equivalent laser power the lifetime of Ru(III) (0.5 ms) (Figure S16) is considerably shorter than in the case of Fe(III) (14 ms). Nevertheless, this is not influencing the solar cell performance, since in both cases regeneration occurs in the µs time scale, leading to unit regeneration efficiency.

To conclude, this study has shown that despite the ps lifetime, C1 gives rise to photoinduced injection into TiO 2 producing a long lived charge separated state (e -(TiO 2 )/Fe(III))surviving several milliseconds in an electrochemically inert electrolyte. Similar dynamics was found in the case of SnO 2 , where, however recombination was faster and performances were generally lower than with TiO 2. Fe(III) could intercept iodide with unitary efficiency a feature which leads, when the photoanode is properly passivated, to the estimation, from APCE, of a quantum injection efficiency close to 50%. This study points out some crucial factors affecting the performance of C1-sensitized DSSCs: i) The nature of the electrolyte and its additives GuNCS and MgI 2 by the way leading to the best performance obtained so far. ii) The recombination of photoinjected electrons with I 3

-due to positive surface potential established upon dye adsorption and the non-optimal screening of the TiO 2 surface from the access of I 3 -. iii) incomplete injection, resulting from kinetic competition of charge transfer with excited state deactivation. In consideration of the excited state energetics, this may result from non-optimal electronic coupling, arising from the symmetric design of C1. Work is in progress to chemically design new FeNHC sensitizers matching these requirements to open the way to efficient iron-based solar devices.

Figure 1 .

 1 Figure 1. J-V curves of TiO2 based C1 sensitized solar cells under AM 1.5G illumination in the presence of I -/I3 -electrolyte. See main text and ESI for details on cell type and electrolyte formulation

Figure 2 .Figure 3 . 3 Please

 233 Figure 2. IPCE of TiO2 based C1 sensitized solar cells collected under short circuit in the presence of I -/I3 -based electrolytes. APCE spectra with BUL/el1 (black) and No BUL/el1 (red) are shown as inset

Figure 4 .

 4 Figure 4. TAS (10 kOhm input impedance) of C1 loaded on TiO2 in contact with el2 deprived of I2. The inset shows the evolution of the transient spectrum during the first 300 µs from excitation (0 µs (black), 20 µs (red), 60 µs (blue), 180 µs (green), 300 µs (purple))

Acknowledgements

The L2CM thanks the French Agence Nationale de la Recherche for a grant (ANR-16-CE07-0013-02) and is grateful to S. Parant for preliminary DSSC measurements. E.M. thanks the Fondo Sociale europeo Programma Operativo (2014-2020) Regione Emilia Romagana for a grant.

Conflicts of interest

There are no conflicts to declare.

Notes and references ‡ TAS carried out on FeNHC sensitized SnO 2 in the µs-ms time scale showed the presence of a recombination component with a time constant of 10 -4 s which persisted also at 350 µJ/cm 2 /pulse. In addition, by comparing with TiO 2 at a comparable excitation fluence and optical density of the sensitized film, the initial amplitude of the FeNHC/SnO 2 signal was reduced by a factor of ca.10, suggesting the presence of an even faster recombination channel at earlier delays (Figure S14).