Electrochemical characterization of a complex FeFe
hydrogenase, the electron-bifurcating Hnd from
Desulfovibrio fructosovorans

Aurore Jacq-Bailly, Martino Benvenuti, Natalie Payne, Arlette Kpebe, Christina Felbeck, Vincent Fourmond, Christophe Léger, Myriam Brugna, Carole Baffert

► To cite this version:

Aurore Jacq-Bailly, Martino Benvenuti, Natalie Payne, Arlette Kpebe, Christina Felbeck, et al. Elec-
trochemical characterization of a complex FeFe hydrogenase, the electron-bifurcating Hnd from Desul-
fovibrio fructosovorans. Frontiers in Chemistry, 2021, 10.3389/fchem.2020.573305. hal-03103487

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# Electrochemical characterization of a complex FeFe hydrogenase, the electron-bifurcating Hnd from *Desulfovibrio fructosovorans*

Aurore Jacq-Bailly¹, Martino Benvenuti¹, Natalie Payne¹, Arlette Kpebe¹,

Christina Felbeck¹, Vincent Fourmond¹, Christophe Léger¹, Myriam

Brugna¹ and Carole Baffert¹,*

¹ Aix Marseille Univ, CNRS, BIP, 31 Chemin Joseph Aiguier, 13402 Marseille cedex
09, France

Correspondence*:

Carole Baffert

cbaffert@imm.cnrs.fr

## 2 ABSTRACT

Hnd, an FeFe hydrogenase from *Desulfovibrio fructosovorans*, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H₂ to both the exergonic reduction of NAD⁺ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases.

In this study, we describe the purification of the enzyme under O₂-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions *via* protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions.

We demonstrate that kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H₂, diffusion and reaction at the active site of CO and O₂) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.

Keywords: direct electrochemistry, FeFe hydrogenase, electron bifurcation, *Desulfovibrio fructosovorans*, inactivation

## 1 ABBREVIATIONS:

FMN: Flavin mononucleotide, NAD: Nicotinamide adenine dinucleotide, NADP: Nicotinamide adenine

dinucleotide phosphate, *bis*PGD: *bis* pyranopterin guanosine dinucleotide, Fd: ferredoxin, SHE: standard

hydrogen electrode
# 2 INTRODUCTION

27

## 2.1 Electron-bifurcating enzymes

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Oxidoreductase enzymes usually catalyze electron transfer between one electron donor and one electron acceptor. Electron-bifurcating enzymes are part of the oxidoreductase family but catalyze the reaction between two electron donors and one electron acceptor or one electron donor and two electron acceptors. More importantly, the reactions with the two electron donors or the two electron acceptors are thermodynamically coupled, one of the reactions being exergonic and the other endergonic. The global reaction being exergonic, the energetic coupling enables an endergonic reaction to occur (Baymann et al., 2018). The first electron-bifurcating enzyme characterized was the cytochrome *bc*₁ complex in which the electron-bifurcating site is a quinone (Mitchell, 1975). More recently, electron bifurcating enzymes were described in which the electron-bifurcation site is a flavin (Herrmann et al., 2008). The common feature of quinones and flavins is their two redox transitions, making them 2-electron centers. A variety of enzymes were described to use the electron bifurcation mechanism for catalysis: electron-transferring flavoprotein (Etf), heterodisulfide reductase/hydrogenase (Hdr-Mvh), NADH-dependent ferredoxin:NADP⁺ oxidoreductase (Nfn) and NADH-dependent FeFe-hydrogenase to name but a few (Peters et al., 2016; Buckel and Thauer, 2018). Electron bifurcation is a mechanism that is emerging as essential for the bioenergetic of many organisms, but this mechanism is still poorly understood, in part due to the low number of model enzymes characterized to date.

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## 2.2 Electron-bifurcating hydrogenases

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NADH-dependent electron-bifurcating hydrogenases are multimeric (tri- or tetrameric) enzymes and are classified A3 according to the hydrogenase classification proposed by Greening *et al.* (Søndergaard et al., 2016; Greening et al., 2016). They are all of FeFe-type and consist of at least one subunit harboring the catalytic H-cluster (the hydrogenase active site, which consists of a [4Fe4S] cluster bound *via* a cysteine to a 2Fe subcluster), a [2Fe2S]-cluster containing subunit and a subunit that contains a flavin (usually FMN) and FeS clusters as well as an NADH binding site. They catalyze the oxidation of H₂ coupled to the reduction of both NAD⁺ and a ferredoxin with a bifurcation mechanism and/or the reduction of proton coupled to oxidation of NADH and a ferredoxin with a confurcation mechanism. So far, electron-bifurcating hydrogenases from five anaerobic bacteria have been purified and characterized (Schuchmann and Mueller, 2012; Wang et al., 2013; Zheng et al., 2014), including the electron-bifurcating hydrogenase HydABC from *Thermotoga maritima* (Schut and Adams, 2009) and HndABCD from *Desulfovibrio fructosovorans* (Kpebe et al., 2018). They have been tested for either electron bifurcation, or electron confurcation or for both (hydrogenase from *Moorella thermoacetica* (Wang et al., 2013)). However, no 3D-structure of an electron bifurcating hydrogenase is available, and the electron pathway and the electron bifurcation site in these enzymes are still controversial subjects. Further characterization of electron bifurcating hydrogenases that could be models of this class of enzyme, will increase the understanding of the overall mechanism of electron bifurcation.

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## 2.3 Electrochemistry of FeFe hydrogenases

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Electrochemical techniques to study hydrogenases are developed as a complement to biochemical and spectroscopic techniques (Pershad et al., 1999). The first electrochemical characterization of an FeFe hydrogenase was published on the HydA hydrogenase from *Megasphaera elsdenii* (Butt et al., 1997; Caserta et al., 2018). Since then, FeFe hydrogenases from several organisms have been studied using protein film voltammetry to determine their catalytic properties: HydAB from *Desulfovibrio desulfuricans*
68 (Parkin et al., 2006; Vincent et al., 2005; Goldet et al., 2009; Rodríguez-Maciá et al., 2018), HydA from 69 Clostridium acetobutylicum (Baffert et al., 2008; Goldet et al., 2009; Baffert et al., 2011, 2012; Kubas 70 et al., 2017; Orain et al., 2015), HydA1 from Chlamydomonas reinhardtii (Stripp et al., 2009; Goldet et al., 71 72 Orain et al., 2015), CpI, CpII and CpIII from Clostridium pasteurianum (Artz et al., 2019), and HydA from 73 Solobacterium moorei (Land et al., 2019). All these FeFe hydrogenases are prototypical A1 monomeric or 74 dimeric enzymes (Søndergaard et al., 2016). Different aspects of the catalytic properties of these enzymes 75 are studied by electrochemical methods: affinity for the substrate (Km), effect of pH on the catalytic 76 properties, kinetics of inhibition by small molecules (CO, O₂, S₂⁻, formaldehyde), kinetics of oxidative 77 and reductive inactivation, and catalytic bias. Only recently was a multimeric Hydrogen Dependent Carbon 78 Dioxide Reductase (HDCR) from Acetobacterium woodii characterized by electrochemistry (Ceccaldi 79 et al., 2017). This hydrogenase is classified A4. It consists of four subunits: the hydrogenase subunit 80 hosting the H-cluster, the formate dehydrogenase subunit hosting the Mo-bisPGD cofactor and two subunits 81 containing several FeS clusters. Electrochemical experiments similar to those developed for prototypic 82 hydrogenases were performed. All these experiments on the different hydrogenases and variants (mutations 83 of specific amino acids) give insight into the catalytic mechanism of FeFe hydrogenases and the molecular 84 determinants of the inactivations. The second and last multimeric hydrogenase characterized by direct 85 electrochemistry belongs to the electron-bifurcating enzyme family (classified A3), the hydrogenase 86 HydABC from Thermotoga maritima (Chongdar et al., 2020). The enzyme is heterologously produced in 87 Escherichia coli and artificially matured with a synthetic diiron cofactor. The isolated hydrogenase catalytic 88 subunit, as well as the complex HydABC, are adsorbed onto an electrode and cyclic voltammograms are 89 recorded at various pH, to show that the isolated hydrogenase subunit and the trimeric complex behave in a 90 similar fashion.

91 Here, we aim at determining if the catalytic and inhibition properties are influenced by the additional 92 subunits present in NADH-dependent electron-bifurcating hydrogenases or by the condition of purification. 93 We study the electron-bifurcating hydrogenase, HndABCD from Desulfovibrio fructosovorans (shortened 94 name Hnd), by biochemical and electrochemical methods. Hnd is homologously produced and purified 95 in a fully matured form (Kpebe et al., 2018). Because Hnd is still active when purified aerobically, we 96 determine how the purification conditions (aerobic vs anaerobic) influence the catalytic behavior of Hnd.

# 3 MATERIAL AND METHODS

## 3.1 Enzyme purification

98 The production and the purification of Hnd hydrogenase from Desulfovibrio fructosovorans under aerobic 99 conditions were previously described (Kpebe et al., 2018). The procedure was modified to maintain 100 anaerobic conditions: all steps were performed in a glove-box (Jacomex, [O₂] ≤ 2 ppm) except the 101 ultra-centrifugation step, for which anaerobiosis is maintained in the tube due to an airtight plug. The 102 cell lysis was performed by sonication (10 cycles of 30 seconds) and the Strep-tagged hydrogenase was 103 purified on a StrepTactin- Superflow (IBA) column (20 mL). Purification was done according to the 104 manufacturer's instructions. For the comparison of the catalytic properties, the bacterial culture was split in 105 two, and purifications of the Hnd hydrogenase were performed in parallel under both anaerobic and aerobic 106 conditions.

## 3.2 Catalytic activity determination

108 All assays were performed at 30 °C and under anaerobic conditions.
### 3.2.1 $H_2$ oxidizing activity with methyl-viologen (MV)

H₂-oxidizing activity measurements were performed in anaerobic quartz cuvettes, under a pressure of 110 H₂ of 1 bar, in 800 µL of a reaction mixture containing 100 mM Tris-HCl pH 8.0, 2 mM dithiothreitol 111 (DTT), and 50 mM methyl-viologen (MV) (Sigma Aldrich) as an artificial electron acceptor. MV reduction 112 was monitored at 604 nm ($ε$= 13,600 M⁻¹·cm⁻¹) using a UV-Vis spectrophotometer Lambda 25 (Perkin 113 Elmer), between 10 and 200 ng of purified Hnd were added to the mixture to start the reaction. One unit of 114 hydrogenase activity corresponds to the uptake of 1 µmol of H₂/min. 115

### 3.2.2 $H_2$-production activity with methyl-viologen (MV)

H₂-production assays were carried out using dithionite-reduced MV (50 mM of MV were reduced with 117 0.1 M sodium dithionite) as electron donor, in anaerobic 7 mL-serum bottles containing 1 mL of a reaction 118 mixture composed of 100 mM Tris-HCl pH 8.0. The gas phase was 100% N₂. H₂ production was measured 119 using gas chromatography (GC) as previously described (Avilan et al., 2018) and the reaction was started 120 by the addition of 0.3 to 1.1 µg of purified Hnd. One unit of hydrogenase activity corresponds to the 121 production of 1 µmol of H₂/min. 122

### 3.2.3 Electron-bifurcating activity for $H_2$ production

Electron-bifurcating (NAD⁺- and Fd-dependent) H₂-oxidizing activity was assayed as described 124 previously (Kpebe et al., 2018): in anaerobic quartz cuvettes, under 1 bar of H₂, in 800 µL-mixture 125 containing 100 mM Tris-HCl pH 8.0, 5 µM of FMN, 5 µM of FAD, and 3 mM NAD⁺ in the presence of 20 126 µM of purified FdxB ferredoxin from *D. fructosovorans*. NAD⁺- and FdxB-reduction were followed 127 simultaneously by recording a full spectrum every 30 s from 300 nm to 800 nm for 1 h, using a 128 Cary 60 (Varian) in a glovebox. NAD⁺- and FdxB-reduction rates were determined at 340 nm and 129 410 nm respectively using the QSoas software (Fourmond, 2016), an open source program available at 130 www.qsoas.org. The specific activity is given in µmol of NADH/min/mg. The absorption coefficients used 131 were: $ε$(NADH) = 6320 M⁻¹·cm⁻¹, $ε$(FdxB<sub>ox</sub>410nm) = 24,000M⁻¹·cm⁻¹ and $ε$(FdxB<sub>red</sub>410 nm) = 12,000 132 M⁻¹·cm⁻¹ (Kpebe et al., 2018). 133

## 3.3 Electrochemical techniques

All electrochemical experiments were carried out with the electrochemical set-up and equipment described 135 in reference (Léger et al., 2004) in a glovebox (Jacomex) filled with N₂. 1 µL of Hnd enzyme solution was 136 mixed with 1 µL of DTT 1M and 8 µL of phosphate buffer pH 7. The final enzyme concentration was 2 to 137 7 µM. The enzyme was adsorbed (1 µL of the previous mix) onto a pyrolytic graphite edge electrode (PGE, 138 surface area ≈ 3 mm²) previously polished with an aqueous alumina slurry (1 µm). The electrochemical 139 cell contained a pH 7 phosphate buffer and was continuously flushed with pure H₂ or with argon. The 140 temperature was regulated to the desired values by circulating water in the double jacket of the cell. For 141 measuring the rates of inhibition by O₂ or CO, a stock of a buffer saturated by 100% O₂ or 1% CO in 142 99% Argon was kept in a capped serum bottle and small aliquots of this solution were injected into the 143 electrochemical cell using gas-tight syringes. The concentrations of O₂ and CO were calculated using 144 the Henry's law constants: 1.25 mM (atm O₂)⁻¹ and 1 mM (atm CO)⁻¹. The change in H₂ solubility is 145 only about 12% between 10 °C and 30 °C (Wilhelm et al., 1977). This variation induces a difference in 146 the potential of H⁺/H₂ Nernst couple of 2 mV. With the surface area (around 5 mm²) of the electrode, 147 the maximum current (limitation by mass transport) is 100 µA. In the experiments of this study, the H₂ 148 oxidation current (maximum 2 µA) is not limited by mass transport (or less than 2%) (Merrouch et al., 149 2017). The data were analyzed using the QSoas software (Fourmond, 2016). The protein film loss was 150
151 included in the data analysis of Figure 4 according to reference(Fourmond et al., 2009). The effect of film
152 loss on cyclic voltammograms is shown in Figure S5.

4 RESULTS

**4.1 Biochemical comparison between aerobically and anaerobically purified Hnd**

We previously reported the biochemical characterization of the Hnd hydrogenase purified under aerobic
conditions (Kpebe et al., 2018). Here, we repeated the same characterization but with the enzyme purified
under anaerobic conditions. Table 1 summarizes the results obtained with the enzyme purified under the
two conditions. The conditions of purification do not have much influence on the properties of Hnd (Km
for methyl-viologen, optimal temperature and pH). If we compare the ratio of H₂ oxidation activity for the
two enzymes (anaerobically- and aerobically-purified Hnd) with methyl-viologen and with physiological
partners, the ratio is higher when considering the electron bifurcation activity. It should be noted that
the activities are lower than those previously reported (Kpebe et al., 2018) because experiments were
performed with enzyme purified from cells grown for a longer period (2 months, Covid19 lockdown period)
and at lower temperature (20 °C), and the enzyme samples were stored one week in liquid nitrogen before
electron-bifurcating activity measurements. We already observed that the enzyme activity decreases quickly
after purification (by a factor of 10) and then stabilizes (Kpebe et al., 2018). However, we checked that
only the specific activity changes upon storage and not the other biochemical and catalytic properties (See
Table S7).

**4.2 Electrochemical characterization of Hnd**

Hnd purified under the two conditions (aerobic and anaerobic) was characterized using electrochemical
methods developed in our laboratory (Del Barrio et al., 2018b; Sensi et al., 2017). As already observed for
the electron-bifurcating trimeric hydrogenase from *Thermotoga maritima* (Chongdar et al., 2020), despite
its multimeric form, the Hnd hydrogenase can transfer electrons directly to or from an electrode, without
the need for redox mediator. However, it not possible to speculate what is the first electron-relay within the
enzyme, i.e. what cofactor interacts with the electrode surface and whether it is unique (several entry points
could be possible) because structural information is not available for any electron-bifurcating hydrogenase.
Because experiments presented in this study were not performed in presence of NAD or ferredoxin, these
catalytic properties are more representative of a non-bifurcation reaction. We characterized Hnd purified
under aerobic and anaerobic conditions, and compared the catalytic and inactivation kinetic properties.

**4.2.1 Oxidative inactivation and catalytic bias**

**4.2.1.1 Oxidative inactivation**

Figure 1 shows catalytic cyclic voltammograms (current as the function of potential) of Hnd adsorbed
at a PGE electrode for three enzyme samples: aerobically-purified Hnd, anaerobically-purified Hnd, and
anaerobically-purified Hnd stored 1 day at 4 °C in a glove-box that contains about 10% dihydrogen. The
three voltammograms show a decrease in the catalytic current at high electrode potential, indicating an
oxidative inactivation that is reversible as shown on the reverse scan by the increase in current when the
potential is decreased. However, the reactivation is not complete, particularly for anaerobically purified
Hnd, as shown by the red voltammogram. The irreversible loss could be due to protein film desorption
and/or irreversible inactivation. We cannot discriminate between these two processes. It should be noted
that at this scan rate, for aerobically-purified Hnd (black line in Fig. 1), the decrease in current occurs at
191 around -0.15 V vs SHE. In the inset of Fig. 1, the cyclic voltammogram of the anaerobically-purified Hnd
192 (red line) shows two inactivation/reactivations (indicated by the arrows) at electrode potential around -0.35
193 V vs SHE and around -0.15 V vs SHE. After one day of storage of the anaerobically-purified enzyme, the
194 shape of the cyclic voltammogram is similar to that obtained with aerobically-purified Hnd (blue and black
195 lines in Fig. 1).

196 **4.2.1.2 Catalytic bias**

197 The catalytic bias (or catalytic preference (Fourmond et al., 2019; Del Barrio et al., 2018b; Sensi et al.,
198 2017)) is defined as the propensity of a catalyst to catalyze a reaction faster in one direction than in the
199 other. For the same positive and negative overpotential, the activity of the enzyme (i.e. the absolute value
200 of the current in electrochemical experiment) must be compared. The catalytic bias is mainly the function
201 of the rate limiting step of the catalyzed reaction. When inactivation happens, it influences the bias. As
202 shown in Figure 1 anaerobically-purified Hnd is biased toward hydrogen production because it inactivates
203 while oxidizing H₂. Because inactivation and catalytic rate constants vary with temperature, the bias can be
204 modulated by changing the temperature, as illustrated in Figure 2. While anaerobically-purified Hnd is
205 biased toward proton reduction at 30 °C, the opposite is true at 10 °C (See data in Table 2 for a chosen
206 overpotential of η= ± 100 mV, the current of the forward scan was considered). The same behavior was
207 obtained for aerobically-purified and anaerobically-purified Hnd (Figure 2, Figure S1 and Table 2). The
208 same behavior was obtained for aerobically- and anaerobically-purified Hnd (Figure 2, Figure S1 and
209 Table 2). It should be noted that the potential range is not the same in Figure 1(-0.56 to +0.04 V) and in
210 Figure 2 (-0.56 to -0.16 V); thus the second inactivation that happened around -0.15 V is not visible in
211 Figure 2.

212 **4.2.2 Reductive inactivation**

213 Hnd hydrogenase also inactivates at low electrode potential. We used the procedure described previously
214 to study this inactivation (Hajj et al., 2014): a chronoamperogram was recorded while the electrode potential
215 was changed in a 3-step-sequence (E₁-E₂-E₁). E₁ and E₂ values were chosen such as no activation nor
216 inactivation occurs at electrode-potential E₁, and such as detectable reductive inactivation takes place at
217 electrode potential E₂. After each step, a cyclic voltammogram (CV) was recorded. The CVs were started at
218 -0.5 V vs SHE and first scan to high potential. As shown in Figure 3, during the step at E= -760 mV vs SHE,
219 45% of the reduction current (i.e. enzyme activity) is lost, while the loss is only 30% after the third step
220 (percentage of the reduction current loss between the end of the first step and the third step) indicating that
221 inactivation is mostly irreversible. No reactivation was detected during this third step but fast inactivation
222 must take place during CV2. The shape of the cyclic voltammogram after low potential inactivation is
223 similar to the initial, thus the catalytic properties (including the bias) were not much affected by the low
224 potential step. The same behavior was obtained for aerobically-purified and anaerobically-purified Hnd
225 (Figure 3 and S2).

226 **4.2.3 Determination of the Km for H₂**

227 We measured the Michaelis constants (Km) by examining how the steady-state H₂ oxidation current
228 depends on H₂ concentration as described in Fourmond et al. (2013). The value indicated in Table 3 for
229 Hnd hydrogenase was determined from three independent experiments. The same value was obtained for
230 Hnd purified either under aerobic or anaerobic conditions.
231 **4.2.4 CO and O₂ inactivation**

232 As the other FeFe hydrogenases, Hnd is inactivated in the presence of gas inhibitors such as CO and O₂.

233 We determined the kinetic constants of these inhibitions and compared their values with those determined

234 for other FeFe hydrogenases previously characterized electrochemically.

235 **4.2.4.1 CO inactivation**

236 CO inactivation was studied using the method described previously (Baffert et al., 2011): small aliquots

237 of CO solution (1% CO and 99% Argon) were added while the current was measured at a constant electrode

238 potential (Figure 4). We determined the kinetic rate constants of CO binding kᵢCO and CO release kₐCO

239 by fitting the model in equation 1. The inactivation rate constants (kᵢCO) were corrected for hydrogen

240 protection (Liebgott et al., 2010). The values are summarized in Table 3, leading to an inhibition constant

241 KᵢCO of 50 nM at 30 °C.

242 **4.2.4.2 O₂ inactivation**

243 Figure 5 shows a chronoamperogram during which an aliquot of O₂-saturated solution is injected at

244 t=150 s, then O₂ is flushed away and its concentration in the electrochemical cell decreases exponentially

245 over time. Before the injection, the decrease in current is due to the oxidative anaerobic inactivation

246 described in section 3.2.1.1. When O₂ is added, the current drops. A small part of this drop is due to

247 direct reduction of O₂ at the electrode, resulting in a negative current. This contribution was taken into

248 account in the modelling. When the dioxygen is flushed out from the solution, a small reactivation could be

249 detected. We used the following model (equation 2) to obtain the modelized curve in Figure 5. Because

250 the initial oxidative anaerobic inactivation is biphasic, the model includes two inactive species of the

251 enzyme (named inactive1 and inactive2) formed during this inactivation. Then a "dead-end" species is

252 formed by O₂ inhibition with a rate that depends on dioxygen concentration. We also tested the model with

253 partly reversible O₂ inactivation, such as described in ref (Orain et al., 2015) but it cannot be fitted to the

254 experimental data. A kinetic constant kᵢO₂ of 2.4 mM O₂⁻¹.s⁻¹ was obtained with the modelization (Table

255 3).

256 Similar values for CO and O₂ inhibition kinetic constants were obtained for Hnd purified either under

257 aerobic or anaerobic conditions.

5 DISCUSSION

258 The conditions of purification have little influence on the enzymatic properties of the Hnd hydrogenase

259 (Km for methyl-viologen, Km for H₂, optimal pH and temperature). The purification conditions slightly

260 influence the activity of the enzyme. The difference in activity between anaerobically- and aerobically-

261 purified Hnd is greater with physiological partners using the electron bifurcation mechanism than with

262 artificial redox partner (MV) (Table 1). These results suggest that aerobic purification could partly damage

263 the enzyme, disrupting the complex. This would explain the decrease in H₂-oxidation activity and the

264 higher impact on the electron bifurcation reaction.

265 The catalytic and inactivation rates, *i.e.* the shape of the cyclic voltammogram in Figure 1, depend

266 on the conditions of the purification of Hnd (aerobic vs anaerobic). One additional inactivation process

267 occurs at lower potential for the anaerobically purified Hnd, and this inactivation disappears after one

268 day of storage (see insert of Figure 1). This indicates the presence of two forms of the enzyme that can

269 interconvert. The presence of the two forms and their interconversion hinder a full characterization of the
270 oxidative inactivation. However, the process appears to be biphasic (see Figure 5 before the addition of
271 dioxygen) with the formation of two different inactive species (named inactive1 and inactive2 in equation
272 2). The molecular difference between the two forms is still unknown, and could be due to a small change
273 in the environment of the active site, as well as a conformational change. Further characterization of the
274 interconversion and the oxidative inactivation is in progress in our laboratory.

275 Hnd is inactivated under very mild oxidative conditions compared to standard prototypic FeFe
276 hydrogenases (Del Barrio et al., 2018a), especially when Hnd is purified under anaerobic conditions.
277 This low potential oxidative inactivation was observed for two other hydrogenases: CpIII from Clostridium
278 pasteurianum (Artz et al., 2019) and CbA5H from Clostridium beijerinckii SM10 (Corrigan et al. (2020)
279 and T. Happe et al., unpublished). This property was attributed in CpIII hydrogenase to the lack of polar
280 residues in the vicinity of the H-cluster inducing a low dielectric permittivity (ε). However, by looking at
281 the amino acids in the vicinity of the H-cluster (See supplementary data S4), this conclusion is not valid for
282 CbA5H hydrogenase and even less for Hnd hydrogenase. Furthermore, considering Hnd, the kinetics of the
283 oxidative inactivation and thus the potential at which it occurs depends on the conditions of purification
284 while the amino acid composition is unchanged. This suggests that small changes around the H-cluster
285 tune the rates of the oxidative inactivation process.

286 The bias depends on the rate of catalysis in either direction considered, but also on the rate of the oxidative
287 inactivation. The two depend on temperature, thus the bias could be influenced by the temperature. As
288 shown in Table 2, the catalytic bias can change within only 10 °C, from being toward H₂-production (at 30
289 °C) to being toward H₂-oxidation (at 20 °C). Such a change has not been reported before with any enzyme.

290 In addition to oxidative inactivation, Hnd also inactivates under reducing condition (Figure 3). The shape
291 of the cyclic voltammogram after low potential inactivation is similar (only small changes are visible) to
292 the initial, unlike the case of HydA1 from C. reinhardtii (Hajj et al., 2014). In the case of HydA1 from C.
293 reinhardtii, the change in the shape of the cyclic voltammogram was attributed to the formation of a form
294 of the enzyme with catalytic activity different from that of the just purified enzyme. In the case of Hnd, the
295 species formed under very reductive conditions is fully inactive or the reactivation is so fast that only the
296 active enzyme is present at the beginning of the scan. Further investigations are needed to fully understand
297 this process and to understand the differences with the other FeFe hydrogenases.

298 The Table 3 compares the kinetic constants and inhibition constant by CO and O₂ of various FeFe
299 hydrogenases, including Hnd. With a value of 0.55 bar, the Km for H₂ is similar to that observed for the
300 other FeFe hydrogenases. It should be noted that our set-up does not allow for a pressure of H₂ greater
301 than one atm., which implies that the large value of Km is only measured with low accuracy. CO is a
302 competitive inhibitor of H₂ oxidation by FeFe hydrogenases and H₂ has a protective effect even if the Km
303 for H₂ is high, so we chose to consider the true inhibition constant and not the apparent inhibition constant
304 (Liebgott et al., 2010). While the reactivation kinetic constant (kₐ) does not differ much from one FeFe
305 hydrogenase to the other (around 0.02 s⁻¹), the inhibition kinetic constant (kᵢ) is very dependent on which
306 hydrogenase is considered (Table 3 and Caserta et al. (2018)). The value obtained for Hnd is similar to that
307 observed for the HydAB hydrogenase from another Desulfovibrio bacterium (D. desulfuricans) (Liebgott
308 et al., 2010; Goldet et al., 2009) but also to that of HDCR from Acetobacterium woodii (Ceccaldi et al.,
309 2017). The low inhibition constant (Kᵢ) of these three hydrogenases is probably not due to their multimeric
310 composition but rather to the CO diffusion kinetics to the H-cluster. However, the fast diffusion of CO into
311 these three hydrogenases could be attributed to a higher flexibility needed for the complex formation as
312 proposed by Marsh and Teichmann (2014).
Hnd retains activity even when it is purified under aerobic conditions because it forms an O₂-protected state named Hox inact (Kpebe et al., 2018). Recent studies showed that in the Hox-inact state, a sulfur atom binds the H-cluster: it is either exogenous sulfur (present in the culture media) in the case of "standard" hydrogenases or the sulfur atom of a cysteine in the case of *Clostridium beijerinckii* (Rodríguez-Maciá et al. (2018); Corrigan et al. (2020) and Happe, communication at the 2019 Hydrogenase conference). We recorded CVs in absence and in presence of Na₂S (Figure S6) and we observed the same effect as that described with *D. desulfuricans* while Na₂S has no effect on CbA5H FeFe-hydrogenase from *Clostridium beijerinckii* (Corrigan et al., 2020). Once it is activated under reducing conditions, it becomes sensitive to dioxygen inhibition as shown in Figure 5, which has been already observed for HydAB from *D. desulfuricans* (Roseboom et al., 2006; Rodríguez-Maciá et al., 2018). Unlike the FeFe hydrogenase CbA5H from *Clostridium beijerinckii* (Morra et al., 2016; Corrigan et al., 2020), Hnd is not converted from the active state back to the Hox inact state when exposed to O₂. Many FeFe hydrogenases can not form Hox inact state and are inhibited by O₂, either irreversibly or partially reversibly when O₂ is flushed away. In the later case, the overall O₂-sensitivity is defined by the effective inhibition rate constant kₐ₈O₂ (Table 3) (Caserta et al., 2018). Hnd data are better fitted to the model in equation 2 with the inhibition by O₂ being irreversible, and only the kinetic constant kₐ₈O₂ can be determined. It could be directly compared to kₐ₈O₂. The sensitivity to O₂ of Hnd is much higher than that of HydA1 from *C. acetobutylicum* and HydA from *M. elsdenii* but similar to that of HydA1 from *C. reinhardtii* and HDCR from *Acetobacterium woodii*. *Desulfovibrio* bacteria can face transient exposure to dioxygen under physiological conditions and indeed possess oxygen reductases (Dolla et al., 2006; Schoeffler et al., 2019). Furthermore, different enzymes from *Desulfovibrio* species were shown to resist exposure to O₂ (pyruvate:ferredoxin oxidoreductase (Vita et al., 2008) and CO-dehydrogenase (Merrouch et al., 2015)). The reversible oxidative inactivation of Hnd could be another mechanism for O₂-protection, under physiological conditions.

## 6 CONCLUSION

Here were reported the full enzymatic characterization, using biochemical and electrochemical methods, of the electron-bifurcating hydrogenase Hnd from *D. fructosovorans* purified either under aerobic or anaerobic conditions. While usually the conditions of purification are not considered (or the purification is only possible under one condition), we show that the conditions of purification could influence the properties of the enzyme: the presence or the absence of air during purification leads to two different forms of the enzyme that can interconvert. These two forms show differences in the catalytic properties, mainly regarding the oxidative inactivation. The anaerobic oxidative inactivation of one of the forms of the enzyme occurs at relatively low potential compared with other characterized hydrogenases, a behavior already observed with two other FeFe hydrogenases: CbA5H from *Clostridium beijerinckii* (Corrigan et al., 2020) and CpIII from *Clostridium pasteurianum* (Artz et al., 2019). The other catalytic properties (Km, CO and O₂ inhibition constant) do not depend on the conditions of purification and are comparable to those of prototypic FeFe hydrogenases already characterized. This indicates that the presence of additional subunits in Hnd complex has little effect on the catalytic and inactivation properties of the hydrogenase. The characterization of the isolated hydrogenase subunit HndD is in progress to verify this hypothesis.

### 6.1 Equations

$$ \text{Active} \underset{k_a^{\text{CO}}}{\overset{k_i^{\text{CO}} \times [\text{CO}]}{\rightleftharpoons}} \text{Inactive} \quad (1) $$
$$ \text{Inactive2} \underset{k_{12}}{\overset{k_{21}}{\rightleftharpoons}} \text{Inactive1} \underset{k_{A1}}{\overset{k_{1A}}{\rightleftharpoons}} \text{Active} \xrightarrow{k_i^{O_2} \times [O_2]} \text{Dead end} \quad (2) $$

351 6.2 Figures

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Cyclic voltammograms of Hnd hydrogenase adsorbed on a PGE electrode; black: aerobically-purified enzyme, red: anaerobically-purified enzyme, blue: anaerobically-purified enzyme, stored 1 day (J+1) anaerobically at 4 °C. The current for the aerobically-purified enzyme (black line) was multiplied by two for the sake of clarity. Insert: zoom in the high potential range. Scan rate: 20 mV/s, T= 30 °C, 1 bar H₂, phosphate buffer pH 7, ω= 3000 rpm</figcaption></figure>

352 6.3 Tables

7 NOMENCLATURE

CONFLICT OF INTEREST STATEMENT

353 The authors declare that the research was conducted in the absence of any commercial or financial

354 relationships that could be construed as a potential conflict of interest.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Cyclic voltammograms of anaerobically-purified Hnd hydrogenase adsorbed on PGE electrode as a function of temperature; blue: 10 °C, black: 20 °C, red: 30 °C. Scan rate: 20 mV/s, 1 bar H₂, phosphate buffer pH 7, ω= 3000 rpm</figcaption></figure>

<table><caption>**Table 1.** Comparison of catalytic properties of Hnd purified either under aerobic or anaerobic conditions. The values are the average of three independent experiments. T= temperature, SA= specific activity. <sup>a</sup> Data from Kpebe et al. (2018).</caption><thead><tr><th>Purification conditions</th><th>Km (MV) (in mM)</th><th>Optimal T (in °C)</th><th>Optimal pH</th><th>SA (MV) H₂ oxidation (in U/mg of enzyme)</th><th>SA (MV) H₂ production (in U/mg of enzyme)</th><th>SA (bifurcation) H₂ oxidation (in U/mg of enzyme)</th></tr></thead><tbody><tr><td>Aerobic</td><td>15 ± 2<sup>a</sup></td><td>55<sup>a</sup></td><td>8<sup>a</sup></td><td>475 ± 40</td><td>64.5 ± 13</td><td>0.21 ± 0.12</td></tr><tr><td>Anaerobic</td><td>13 ± 2</td><td>55</td><td>8</td><td>740 ± 45</td><td>53.5 ± 5</td><td>0.72 ± 0.3</td></tr><tr><td>Ratio (anaerobic/ aerobic)</td><td></td><td></td><td></td><td>1.56</td><td>0.83</td><td>3.43</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

AB, MB, NP, AK and CB purified the proteins and performed experiments. CF, VF and CB analyzed the data. CL, MB and CB instigated the research. All the authors wrote the manuscript.
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Reductive inactivation of aerobically-purified Hnd hydrogenase adsorbed on PGE electrode. Main: chronoamperogram, *E*= -510 mV vs SHE for t<250 s and t>700 s and *E*= -760 mV vs SHE for 250<t<700 s. Insert: cyclic voltammograms recorded after the first potential step (CV1, black line), after the second step (CV2, blue line) and after the last step (CV3, red line). The background current was subtracted. T=30 °C, 1 bar H₂, phosphate buffer pH 7, *ω*= 3000 rpm.</figcaption></figure>

<table><caption>**Table 2.** Catalytic bias data extracted from Figure 2 and Figure S1, for an overpotential *η*= ± 100mV.</caption><thead><tr><th rowspan="2">Temperature</th><th colspan="2">I(η=100mV)</th><th colspan="2">Bias</th></tr><tr><th>(in μA)</th><th>(in μA)</th><th>I(η=100mV)/|I(η=-100mV)|</th><th>anaerobic</th><th>aerobic</th></tr></thead><tbody><tr><td>30°C</td><td>0.20</td><td>-0.45</td><td>0.44</td><td></td><td>0.21</td></tr><tr><td>20°C</td><td>0.50</td><td>-0.30</td><td>1.67</td><td></td><td>1.25</td></tr><tr><td>10°C</td><td>0.55</td><td>-0.20</td><td>2.75</td><td></td><td>1.93</td></tr></tbody></table>

## FUNDING

NP is funded by Doc2amu program which has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No713750. Also, Doc2amu has been carried out with the financial support of the Regional Council of Provence-Alpes-Cote d'Azur and with the financial support of the A*MIDEX (nº ANR-11-IDEX-0001-02), funded by the Investissements d'Avenir project funded by the French Government, managed by the French National Research Agency (ANR). This work was funded by the French National Research Agency (ANR-12-BS08-0014, ANR-14-CE05-0010) and by the A*MIDEX funded by the Investissements d'Avenir project (ANR-11-IDEX-0001-02).
<figure><img src="image_7.png" /><figcaption>**Figure 4.** Inactivation by CO of aerobically-purified Hnd hydrogenase adsorbed on PGE electrode. The black line corresponds to experimental data and the dashed red line corresponds to best fit of the model in equation 1. [CO]= 288 nM, 192 nM, 96 nM, 288 nM and 192 nM injected at respectively t= 100 s, 500 s, 850 s, 1220 s and 1540 s; E= -360 mV vs SHE, T=30 °C, 1 bar H₂, phosphate buffer pH 7, ω= 3000 rpm.</figcaption></figure>

ACKNOWLEDGMENTS

The authors thank I. Meynial-Salles and P. Soucaille for kindly providing the plasmid pthl-Fd-LL-C-Tag.

The authors are part of the French bioinorganic chemistry network (www. frenchbic.cnrs.fr).

SUPPLEMENTAL DATA

S1: Cyclic voltammograms of aerobically-purified Hnd hydrogenase adsorbed on PGE electrode as

a function of temperature; S2: Reductive inactivation of anaerobically and aerobically-purified Hnd

hydrogenases; S3: Parameters used for the modelization of Figure 5; S4: Amino-acids in the vicinity of

the H-cluster for several hydrogenases; S5: successive cyclic voltammograms of aerobically-purified Hnd

hydrogenase; S6: Cyclic voltammograms of aerobically-purified Hnd hydrogenase without and with 1mM

Na₂S; S7: Specific activity and biochemical and catalytic properties of different enzyme preparation.

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