# Turning the FeFe hydrogenase from Clostridium beijerinckii into an efficient H2 oxidation catalyst using a redox-active matrix

Dawit T Filmon, Jan Jaenecke, Martin Winkler, Vincent Fourmond, Christophe Léger, Nicolas Plumeré

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## To cite this version:

Dawit T Filmon, Jan Jaenecke, Martin Winkler, Vincent Fourmond, Christophe Léger, et al. Turning the FeFe hydrogenase from Clostridium beijerinckii into an efficient H2 oxidation catalyst using a redox-active matrix. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122 (41), pp.e2514698122. 10.1073/pnas.2514698122. hal-05306583

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# Turning the FeFe hydrogenase from *Clostridium beijerinckii* into an efficient H₂ oxidation catalyst using a redox-active matrix

Dawit T. Filmon<sup>a,1</sup> <sup>ID</sup>, Jan Jaenecke<sup>a,1</sup>, Martin Winkler<sup>a</sup> <sup>ID</sup>, Vincent Fourmond<sup>b</sup> <sup>ID</sup>, Christophe Léger<sup>b,2</sup> <sup>ID</sup>, and Nicolas Plumeré<sup>a,2</sup> <sup>ID</sup>

Affiliations are included on p. 7.

Edited by Marcetta Darensbourg, Texas A&M University-College Station, College Station, TX; received June 6, 2025; accepted September 11, 2025

Hydrogenases are Nature's sustainable and efficient catalysts for the conversion between H⁺ and H₂. The obstacles that prevented their use as H₂ oxidation catalysts in fuel cells are being removed one by one, thanks to the continuous discovery of hydrogenases that have unexpected catalytic properties, the development of new methods for their scalable production and matrices that protect them. Obtaining an efficient biohybrid electrode that is scalable and robust under a large range of experimental conditions is still challenging. The FeFe hydrogenase of *Clostridium beijerinckii* is a very active catalyst of H₂ evolution and can be handled under O₂, and its production can potentially be scaled up. However, it was believed that it cannot be used for H₂ oxidation, as it is easily oxidized to an O₂-stable but inactive state. Here, we show that when the enzyme is embedded into a redox-active film whose reduction potential is finely tuned to the equilibrium potential of the H⁺/H₂ couple, the potential that the enzyme experiences can be buffered in a sharp window that is actually compatible with enzyme-catalyzed H₂ oxidation and prevents anaerobic inactivation. This leads us to provide the demonstration of an FeFe hydrogenase-based system that can be used for H₂ oxidation and that can be repeatedly exposed to O₂ during and between operational cycles.

hydrogenase

oxygen resistance

hydrogen oxidation

redox-active films

catalyst reactivation

The use of hydrogenases as alternatives to precious metal catalysts (1) for H₂ oxidation in fuel cells has been a very active field of research (2–6). The main challenge in using hydrogenases comes from their sensitivity to oxidative (anaerobic or aerobic) conditions (1). There are various ways around this issue. One is to embed the fragile enzyme into a protective matrix (7, 8). The other is to explore the biodiversity to identify, produce, characterize, and eventually use enzymes that are particularly robust (9).

Two main families of hydrogenases have been considered, NiFe and FeFe hydrogenases (the names of these enzymes refer to the metal content of their active site). Some hydrogenases are described as “O₂-sensitive” because they quickly react with O₂ to produce inactive states. This inactivation may be irreversible (the active site is destroyed, as occurs with most FeFe hydrogenases) or reversible. If the activity is recovered upon reduction, the enzyme is referred to as “O₂-stable”; this is the case of all standard NiFe hydrogenases and the FeFe hydrogenase from *Clostridium beijerinckii*. If this reactivation is fast even above the H⁺/H₂ equilibrium potential, the enzyme can oxidize H₂ in the presence of O₂ and is called “O₂-resistant.” This is the case of the NiFe hydrogenases from the phylogenetic group 1d (9, 10).

Because the presence of O₂ is unavoidable in H₂/O₂ fuel cells, early efforts focused on using these O₂-resistant NiFe hydrogenases (11–14). The subclass of NiFeSe hydrogenases has also been considered because these O₂-stable enzymes reactivate relatively quickly upon reduction (15, 16). However, the biosynthesis of NiFe hydrogenases involves a very heavy biological machinery, and despite recent advances (17), they cannot be produced in large quantities, limiting their potential as a scalable alternative to precious metal catalysts.

In contrast, recent progress in the artificial maturation of FeFe hydrogenases (18), whose active site is a dinuclear cluster of Fe (Fig. 1), facilitated the production of some of them by heterologous production of the apo-enzymes (devoid of dinuclear cluster) and their activation upon subsequent incorporation (19, 20) of a synthetically produced (21) diiron complex. All steps are potentially compatible with a large-scale production of the enzyme. However, FeFe hydrogenases are deemed very O₂-sensitive: none discovered so far can oxidize H₂ in the presence of O₂ under conditions of direct electron transfer (DET), and in the case of most FeFe hydrogenase characterized so far, the binding of O₂ to their active site leads to irreversible degradation in a multistep process (22–24).

## Significance

Hydrogenases are large and efficient metalloenzymes that catalyze the conversion between protons and hydrogen, with an inorganic active site that coordinates cheap transition metals. They have long been considered to replace precious metals as catalysts in fuel cells, but despite their diversity in the microbial world, none of the hydrogenases identified so far meet all the requirements. The FeFe hydrogenase from the bacterium *Clostridium beijerinckii* comes close: Its production can be scaled up and it can be exposed to air without being destroyed. However, its protection from oxidative damage also prevents H₂ oxidation catalysis. We show that the enzyme can actually oxidize H₂, even in the presence of O₂, when it is embedded in a finely tuned redox environment.

Author contributions: C.L. and N.P. designed research; D.T.F., J.J., M.W., V.F., and C.L. performed research; D.T.F., J.J., M.W., V.F., and C.L. analyzed data; and D.T.F., J.J., C.L., and N.P. wrote the paper.

Competing interest statement: All authors are inventors on a European patent application (EP25305533.9), filed on April 14, 2025, with the title "Enzymatic catalysis of hydrogen oxidation and reduction," submitted by Technical University of Munich that covers the electroenzymatic catalysis of hydrogen oxidation and reduction.

This article is a PNAS Direct Submission.

Copyright © 2025 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

¹D.T.F. and J.J. contributed equally to this work.

²To whom correspondence may be addressed. Email: leger@imm.cnrs.fr or nicolas.plumere@tum.de.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2514698122/-/DCSupplemental.

Published October 9, 2025.
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Potential window for H₂ evolution, H₂ oxidation, and inactivation of the FeFe hydrogenase from *C. bejerinckii* (CbA5H) under conditions of DET with electrodes and for the same enzyme when embedded in redox-active films made of dendrimers DV1 and DV2 (mediated electron transfer, MET). Under DET, the catalytic current for H₂ oxidation is lost at potentials more positive than -0.3 V vs. Standard Hydrogen Electrode (SHE) (red dashed line). DV1 fails to support H₂ oxidation because its redox potential lies above the potential where the enzyme inactivates. In contrast, DV2 has a redox potential that aligns with the narrow potential window in which CbA5H remains active for H₂ oxidation. The redox buffering effect of DV2 maintains the hydrogenase in its active form regardless of the applied potential at the electrode, which extends its catalytic activity for H₂ oxidation far beyond E<sub>inact</sub>.</figcaption></figure>

The use of these O₂-sensitive FeFe hydrogenases is enabled by embedding them in redox-active matrices for protection from O₂ under aerobic turnover conditions for H₂ oxidation (7). However, this protection from O₂ is strictly dependent on the presence of H₂, and thus it is effective only under operational conditions, but not during shutdown or storage. Some FeFe hydrogenases can be protected via inhibition by exogenous sulfide (25, 26) because sulfide and O₂ compete for binding to the same coordination site (Fig. 1B) but this is only useful for electrode preparation and handling before catalysis. The redox-driven release of the sulfide ligand activates the enzyme and makes it O₂ sensitive (27, 28).

The recently discovered FeFe hydrogenase from *C. bejerinckii* (CbA5H) is unusual because the sulfide ligand, whose binding to the active site Fe ion can prevent O₂ attack, is intrinsically present as a cysteine side chain borne by a flexible loop (Fig. 1C) (19, 29–31). The Fe–cysteine bond is tight under oxidative conditions, e.g., in the presence of O₂ or when the enzyme is wired to an electrode whose potential is moderately high (>-0.3 V vs. SHE, pH 7.0). Based on the data in ref. 29, we estimate an inactivation potential E<sub>inact</sub> ≈ -250 mV vs. SHE at pH 7 (or +170 mV vs. RHE) from the value of the electrode potential where the inactivation and reactivation rate constants are equal. The distal Fe is also the H₂ binding site, and the oxidation (upon exposure to air or mildly oxidative potentials in DET experiments) that triggers this protection also makes the enzyme inactive. This restricts the potential range for enzyme-catalyzed H₂ oxidation to a narrow window, strongly limiting its catalytic performance and initially suggesting that the enzyme was unsuitable for H₂ oxidation (19, 29–31).

Here, we show that the O₂-stable FeFe hydrogenase CbA5H can actually sustain H₂ oxidation under very oxidizing conditions, provided that it is embedded into a redox-active film, but only on condition that the redox properties of the latter are finely tuned to the H⁺/H₂ equilibrium potential (E<sub>eq</sub>). The polymer matrix also makes the system O₂-resistant. We could therefore design a hybrid H₂ oxidizing electrode based on a scalable FeFe hydrogenase that remains active under all conditions, both aerobic and anaerobic, both in the presence and in the absence of H₂.

## Results

We used previously reported viologen-functionalized dendrimers to immobilize hydrogenases within redox-active films on electrode surfaces. Although polymeric backbones are more commonly used for hydrogenase immobilization, we chose the dendrimeric backbones for their ability to form thin, homogeneous films, which facilitate the quantitative analysis of the resulting current response (32, 33). The viologen-modified dendrimers (Fig. 2A) were based on either alkylated 4,4'-bipyridine (DV1) or on alkylated 2,2'-bipyridine (DV2) bearing thioacetate groups to enable crosslinking for film formation (32, 34). The redox potentials of 2,2'-viologens are generally more negative than those of 4,4'-viologens, due to ring-induced strain that destabilizes the radical cation formed upon one-electron reduction (35). We used cyclic voltammetry of films based on DV1 and DV2 dendrimers to determine their redox potentials, yielding values of -0.22 and -0.40 V vs. SHE, respectively (Fig. 2B). In this figure, the cyclic voltammogram (CV) of DV1 exhibits characteristics intermediate between diffusion-controlled and surface-confined behavior (36), whereas the CV of DV2 displays a more pronounced diffusion-like behavior, indicating differences in film thicknesses between the two systems.

We recorded CVs for the hydrogenase from CbA5H under two different electron transfer conditions: DET (Fig. 2C, red), achieved by simple enzyme adsorption onto the electrode, and mediated electron transfer (MET, Fig. 2C, green), where the enzyme is embedded in a film of DV2. Both measurements were conducted with the electrodes rotating in a solution saturated with H₂, under anaerobic conditions. In both DET and MET cases, H₂ evolution is observed as a negative current below the equilibrium potential (E<sub>eq</sub> = -0.42 V vs. SHE at pH 7), and H₂ oxidation occurs above this value. However, under DET conditions, the enzyme inactivates at potentials above -0.3 V as indicated by a drop in H₂ oxidation current as reported by several groups (19, 30, 37). The subsequent recovery of the reductive current in the return sweep to low potential shows that this inactivation is reversible. It is attributed to the binding of the cysteine to the active site (Fig. 1) under mildly oxidizing conditions (29). In contrast, under
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Prevention of high-potential inactivation of CbA5H via MET. (A) Chemical structure of the viologen moieties in the dendrimers DV1 and DV2. (B) CVs of glassy carbon electrodes modified with DV1 (blue) and DV2 (green) at pH 7.0. DV1 (2.8 µg) and DV2 (0.57 µg) were deposited by drop-casting on the electrodes. The currents are normalized by the maximal positive current. (C) CVs of CbA5H under DET conditions (red) and embedded in redox hydrogel films of DV2 (green) at pH 7.0. The current is normalized by the maximal positive current. (D) Chronoamperometry (CA) of CbA5H under DET conditions (red) and embedded in films of DV2 (green) at -0.6 and +0.2 V vs. SHE at pH 7.0. The catalytic current is normalized by the reductive current measured at *t* = 2 min. CVs of CbA5H (0.16 nmol) embedded in films of DV2 (0.57 µg) (E) or of DV1 (2.8 µg) (F) at pH 8.8, 7.0, and 6.0. All MET measurements were conducted on a glassy carbon electrode (*d* = 3 mm) in Tris-HCl/Citrate buffer (50 mM) with KCl (100 mM). All DET measurements were conducted on a pyrolytic graphite edge electrode (4 × 4 mm²) in phosphate buffer (100 mM) with NaCl (100 mM). All experiments were conducted at *v* = 10 mV s⁻¹, *T* = 20 °C, *ω* = 2,000 rpm, 100% H₂ atmosphere. The nonnormalized CVs of C and D are shown in *SI Appendix*, Figs. S1 and S2. The SD of *E* and *F* are shown in *SI Appendix*, Figs. S3 and S4, respectively. Limitations of H₂ mass transport were excluded via control experiments showing that the catalytic current is independent of the rotation rate at high electrode rotation rates (*SI Appendix*, Fig. S5), and of H₂ concentration at high H₂ concentrations (*SI Appendix*, Fig. S6). CVs of CbA5H embedded in DV2 with different amounts show a linear increase in catalytic current, which is consistent with the behavior of thin films (*SI Appendix*, Fig. S7) (34).</figcaption></figure>

conditions of MET, the H₂ oxidation current is stable even at potentials up to 0.2 V vs. SHE (green in Fig. 2C). The observed plateau current across the potential range between -0.3 and 0.2 V vs. SHE is characteristic of catalysis by redox enzymes embedded in redox-active films on rotating electrodes, operating under conditions that are free from inactivation (34, 38). This difference in response to oxidative potential is further confirmed by chronoaamperometry (CA) experiments (Fig. 2D), where the potential is stepped between -0.6 V and +0.2 V vs. SHE: Under MET conditions, both H₂ oxidation and evolution are observed (green in Fig. 2D), while under DET conditions, only H₂ evolution is visible (red in Fig. 2D).

To determine whether high-potential-induced inactivation can also occur under conditions of MET, we repeated the MET experiment using the more oxidizing 4,4'-viologen modified redox dendrimer (DV1, Fig. 2F) whose potential is 0.18 V more positive

than that of DV2 (E⁰ = -0.22 V vs. SHE, Fig. 2B). Fig. 2 E and F compare CVs of Cb5AH embedded into films of DV2 (panel E) and DV1 (panel F) at various pH values. In the case of DV2, the pH values of 6, 7, and 8.8 correspond to relatively small E⁰ - E(H⁺/H₂) values (-40, 20, and 120 mV, respectively), and the CVs show sustained H₂ oxidation without signs of inactivation. The catalytic bias shifts toward H₂ oxidation with increasing pH as described previously (34) (Fig. 2E). In contrast, for DV1, the corresponding E⁰ - E(H⁺/H₂) values are much higher (140, 200, and 300 mV), and all CVs exhibit anaerobic inactivation (Fig. 2F). At the higher pH values, the inactivation becomes more pronounced as indicated by the steeper slope in the current decrease at higher potentials (Fig. 2F). This is attributed to the E⁰ of the viologen being pH independent, whereas the kinetics of inactivation is pH dependent (29). At neutral and acidic pHs, the inactivation is slightly less pronounced, however, the catalysis is shifted
toward H₂ uptake due to the mismatch of the mediator potential and catalysis potential (34).

To better understand the effect of changing the potential of the
viologen (E⁰), we calculated its Nernst potential (E_v) within the
redox-active films using the concentration profiles of oxidized and
reduced viologen, which were obtained by solving a kinetic reac-
tion-diffusion model. Unlike in our previous work (39-41), this
model assumes bidirectional H⁺/H₂ conversion by the enzyme
(see details in SI Appendix). The simulations were performed using
parameters relevant to the experiments carried out at pH 6, 7, and
8.8 (shown in Fig. 2 E and F), considering the E⁰ values of DV1
and DV2 relative to the H⁺/H₂ equilibrium potential, keeping all
other parameters constant. The results of the simulation for pH
7 are shown in Fig. 3, and those for pH 6 and 8.8 are shown in
SI Appendix, Figs. S8 and S9, respectively.

Panels A and C show the steady-state anaerobic concentration
profiles. The concentration profiles of H₂ (gray), oxidized viologen
(pink), and reduced viologen (blue), along with the Nernst poten-
tial of the viologen (dark green and right Y-axis), are shown as a
function of the normalized distance from the electrode (ξ = 0 at
the electrode surface to ξ = 1 at the film/solution interface), under
H₂ oxidation conditions (i.e., high electrode potential) and in the
absence (left column) or presence (right column) of O₂. In all
cases, the simulations reveal a slight depletion of H₂ within the
film (the H₂ concentration is lower than in the bulk of the solu-
tion), and the accumulation of oxidized viologen near the elec-
trode surface. The Nernst potential of the viologen matches the

electrode potential only in a tiny region close to the electrode.
Away from the electrode (ξ < 0.2), where catalytic H₂ oxidation
occurs, it decreases to a value that equates roughly the E⁰ of the
viologen (marked by horizontal dashed green lines), and further
from the electrode (0.2 < ξ < 1), it equilibrates with that of the
H⁺/H₂ couple, irrespective of the standard potential of the violo-
gen, so that E_v tends to 0 vs. RHE.

Fig. 3 also indicates with a horizontal dashed gray line the approximate value of the inactivation potential of CbA5H (≈0.17 V vs. RHE). The effect of using one redox-active dendrimer or the other is clear from Fig. 3: The Nernst potential that is experienced by the enzyme in the region close to the electrode where H₂ oxidation occurs is higher or lower (DV1 and DV2, respectively) than the inactivation potential of CbA5H. This observation is therefore consistent with the inactivation not occurring when the enzyme is embedded in DV2 (E⁰ = -0.4 V vs. SHE, Fig. 2E).

The same is observed over a range of pH from 6 to 8.8 (see the
simulations in panels *A* and *C* of *SI Appendix*, Figs. S8 and S9,
respectively). The potential of DV2 remains below *E*<sub>inact</sub> which
explains the sustained H<sub>2</sub> oxidation in this pH range (Fig. 2E). In
contrast, DV1, is higher or near *E*<sub>inact</sub> in this pH range, leading to
deactivation (Fig. 2F).

Having shown that the hydrogenase from CbA5H can be pro-
tected from oxidative, anaerobic inactivation under conditions of
MET, we turn to the protection from aerobic inactivation. To
assess the ability of redox-active films to protect the enzyme from
O₂, we incorporated CbA5H into the DV2 dendrimer system and

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Simulation of the distribution of the different species within the films in the presence and absence of O₂. Concentration profiles of Vox (pink), Vred (blue), H₂ (gray), O₂ (red), and inactive enzyme (black), and the viologen Nernst potential vs. Reversible Hydrogen Electrode (RHE) (dark green and Right Y-axis) (calculated using the Nernst equation from the ratio Vox/Vred) as a function of the normalized distance from the electrode, with viologen potentials matching those of DV1 (Top, panels A and B) and DV2 (Bottom, panels C and D), calculated under oxidizing, anaerobic conditions (Left, panels A and C) and aerobic conditions (Right, panels B and D). The anaerobic concentration profiles are in the steady-state, the aerobic concentration profiles are snapshots calculated at the same time after exposure to O₂. The standard potentials of the viologens and the potential where the enzyme inactivates (29) are constant parameters and shown as horizontal dashed lines (they do not depend on distance, unlike the Nernst potential). See SI for details about the simulations.</figcaption></figure>
into a previously reported 2,2'-viologen-modified polyvinyl alcohol (PV2) matrix (34). PV2 bears the same viologen moiety as DV2 and displays a similar redox potential of -0.431 ± 0.007 V vs. SHE. PV2 was selected as an additional matrix for the aerobic experiments because it enables the formation of more stable films compared to DV2, possibly due to the larger molecular weight of its building blocks. This enhanced stability makes PV2 well-suited for studying the resistance of CbA5H films to O₂ upon prolonged exposure. Under anaerobic conditions, CbA5H embedded in PV2 films show sustained currents for H₂ oxidation (SI Appendix, Fig. S11) comparable to those observed with DV2 (Fig. 2E). This demonstrates that PV2, like DV2, effectively protects CbA5H from high-potential inactivation, which is expected since PV2 and DV2 have similar redox potentials.

**Stability in Air in the Absence of H₂.** CVs of DV2/CbA5H-modified electrodes before (black) and after (gray) exposure to air for 3 h show a minor decrease in the H₂ oxidation current (Fig. 4A), demonstrating that the enzyme embedded in the redox-active film remains O₂-stable (29, 31). The catalytic current retains 74 ± 15% of its initial value even after 18 h of exposure to ambient air following an initial activation cycle under H₂ (green in Fig. 4B). This stability is attributed to CbA5H's intrinsic protection mechanism, which relies on reversible inactivation of its active site under oxidative conditions. In contrast, the sulfide-protected

FeFe hydrogenase from *Desulfovibrio desulfuricans* only tolerates air exposure prior to activation. Once activated by H₂, subsequent exposure to O₂ leads to rapid and irreversible inactivation. After just 1 h of air exposure, the H₂ oxidation current drops below 11%, and complete loss of activity is observed after 3 h (gray in Fig. 4B). The capacity of CbA5H/DV2 modified electrodes to withstand repeated activations and air exposures under conditions of open circuit were also evaluated by CV (Fig. 4C). The decrease in H₂ oxidation current is about 20% per exposure, resulting in an overall loss of activity of about 65% after four cycles. The same behavior is observed for H₂ production. This overall trend is consistent with previous findings from solution-phase assays by Rutz et al. (30), who reported a 75% loss in catalytic activity of CbA5H for H₂ production after two exposures to air. The similar losses observed both in redox-active films for H₂ oxidation and H₂ evolution and in solution-phase H₂ evolution assays suggest that the decline in activity primarily arises from enzyme inactivation rather than limitations of the immobilization matrix.

**Protection from O₂ during H₂ Oxidation.** The CA experiment in Fig. 4D shows that the H₂ oxidation current recorded with CbA5H in PV2 under a mixed H₂ and O₂ gas feed is barely affected by O₂. As observed before with the O₂-sensitive FeFe hydrogenase from *Chlamydomonas reinhardtii*, the decrease in current that results from exposure to O₂ is small and reversible

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Prevention of O₂-induced inactivation of CbA5H embedded in viologen-modified films. (A) CVs of CbA5H (0.16 nmol) embedded in DV2 (0.57 µg) films in Tris-HCl/Citrate buffer (50 mM) with KCl (100 mM) saturated with H₂ (100%) at pH 7.0 before and after 3 h of exposure to air at 5 °C. (B) Residual current for H₂ oxidation obtained from CVs of redox-active films containing either CbA5H or sulfide-protected DdHydAB after operating the electrode for H₂ oxidation followed by exposure to air for different periods of time. The corresponding CVs of DdHydAB embedded in DV2 before and after the exposure to air are shown in SI Appendix, Fig. S10 [note that the protection of DdHydAB by sulfide is only operational before the film is used for catalysis (27, 28)]. (C) Residual H₂ oxidation currents obtained from CVs of the CbA5H/DV2-modified electrode after repeated cycles of H₂ oxidation followed by O₂-exposure (at 5 °C for 1 h). The currents are normalized with the values obtained for the first CV before exposure to O₂. (D) CAs of CbA5H (0.16 nmol) in PV2 (1.13 µg) at -0.2 V vs. SHE with a 95:5 gas feed of H₂:N₂ (nonshaded area) and H₂:O₂ (gray area). Unless specified otherwise, all experiments were conducted on a glassy carbon electrode (d = 3 mm), v = 10 mV s⁻¹, T = 20 °C, ω = 2,000 rpm, 100% H₂ atmosphere.</figcaption></figure>
(7). The reason is understood by examining the concentration profiles in the *Right* column of Fig. 3 and *SI Appendix, Figs. S8 and S9*). The penetration of O₂ in the film (red curve) inactivates the enzyme molecules that are present in the outer part of the film (black concentration profile, labeled E₁, where “1” is short for “inactive”), and oxidizes the viologen (pink), which progresses inward due to charge hopping between neighboring viologens (in an “apparent” diffusion process) (42). The oxidized viologen in turn, oxidizes the active enzyme, which oxidizes H₂. This reaction regenerates the reduced viologen (blue), which shuttles the electrons outward via hopping and reacts with the incoming O₂, impeding its penetration into the inner layers of the film (40).

The simulations show that the O₂ penetration and the progress of the front of inactive enzyme is faster with DV2 than with DV1. This is because the rate of progress of the front depends on the concentration of reduced viologen (see e.g., equation 3 in ref. 35). The ratio V<sub>ox</sub>/V<sub>red</sub> equilibrates with the H⁺/H₂ couple in the bulk of the film. Switching from DV1 to DV2 corresponds to a decrease in E⁰, which leads to a decrease in the equilibrium concentration of reduced viologen, thus making the front move faster. The simulations for both DV1 and DV2 also show that catalysis for current generation is confined to the region near the electrode which has not been exposed to O₂. This implies that the (reversible) inactivation of the enzyme in the outer layer of the film does not impact the H₂ oxidation current and that the initial current is fully recovered when O₂ is removed (40).

Since the protection from O₂ provided by the redox-active film is entirely dependent on H₂ oxidation in the outer part of the film, the enzyme should inactivate entirely if the film is exposed to O₂ in the absence of H₂. However, due to the intrinsic air stability of the enzyme (that results from the binding of the cysteine to the active site), the hydrogenase remains protected not only during aerobic and anaerobic H₂ oxidation catalysis but also during storage between operational cycles and idle periods associated with intermittent use.

## Discussion

The discovery of a FeFe hydrogenase that retains H₂-evolving activity upon repeated exposure to air came as a surprise (31), since all previously identified FeFe hydrogenases are O₂-labile (that is, their active site is irreversibly damaged upon exposure to O₂) (9). Compared to NiFe hydrogenases, which have been preferred for most applications so far, the advantage of using a FeFe hydrogenase is that their production can be scaled up by using an artificial maturation procedure that involves the separated large-scale productions of the apo-enzyme and its cofactor and their reaction to reconstitute the holo enzyme with full activity (18, 43).

The enzyme from *C. beijerinckii* (31) is one of only two FeFe hydrogenases identified so far that are air-stable. Happe et al. have indeed recently identified and characterized a second air-stable FeFe hydrogenase that is phylogenetically distinct from CbA5H (44). Despite their evolutionary differences, both enzymes share a common structural feature responsible for their oxygen tolerance: the coordination of a nearby cysteine side-chain to the active site iron atom (29, 44, 45).

This protection makes the enzymes easy to handle in air, but it has a downside: It also inactivates the enzyme under exposure to O₂ or even mildly oxidizing, anaerobic conditions, so that the enzyme cannot oxidize H₂ in air (unlike O₂-tolerant NiFe hydrogenases) (9). Morra et al. did use CbA5H for anaerobic H₂-driven NADPH regeneration, but the turnover frequency in the H₂ oxidation direction was indeed very low (46). It therefore seems that because of the tradeoff between O₂-stability and activity, the

enzyme from *C. beijerinckii* cannot be very useful for H₂ oxidation (19, 29–31). And yet, we demonstrated in this paper that this enzyme can actually be used to continuously oxidize H₂, even in the presence of O₂, on condition that it is embedded in a redox-active matrix with a finely tuned redox potential. The matrix prevents oxidative inactivation by maintaining the Nernst potential within a narrow range, high enough to oxidize the enzyme, which oxidizes H₂, but below the inactivation potential of the enzyme. As shown in the *Left* column of Fig. 3, when the electrode potential is high, the viologen Nernst potential that is experienced by the enzyme matches the electrode potential in only a small region near the electrode surface, then it decreases to the standard potential of the viologen in the reaction layer, and finally to the H⁺/H₂ equilibrium potential in the main part of the film (Fig. 3, *Left* column). The effect of the mediator is therefore to buffer the potential of the film at a value close to its standard potential. The latter can be adjusted by synthetic engineering (34). The range of useful viologen standard potentials is up to ≈ -0.3 V, at pH 7. PV2 and DV2 (E⁰ of -0.4 and -0.43 V vs. SHE, respectively) fall within this range and effectively preserve catalytic activity, unlike DV1, which has a significantly higher E⁰ leading to oxidative inactivation of the enzyme (Fig. 2D).

The intrinsic protection of the enzyme from O₂, which involves a flexible cysteine residue and movement of an internal loop (29), remains functional even when the enzyme is embedded in the redox-active matrix (Fig. 4 A–C), as this conformational change is not hindered by the immobilization. This self-protection of the enzyme is maintained efficiently during long-time exposure to ambient air without the need for any treatment (no addition of extrinsic ligand or applied potential). This protection is not perfect: The delayed response to O₂ attack (29) results in a fraction of the enzyme being irreversibly damaged after each exposure to O₂. Therefore, repeated exposure to air gradually reduces the catalytic activity. This behavior is reminiscent of the results of solution assay experiments conducted with CbA5H, where repeated exposure to air led to near-complete activity loss after four cycles. This observation confirms that the current loss that we see in Fig. 4C is due to the enzyme itself, not to the entrapment of the enzyme in the redox-active film compromising its intrinsic stability. Efforts to enhance the stability of CbA5H through mutagenesis have been successful (30).

Under conditions of H₂ oxidation catalysis in the presence of O₂, the general mechanism for protection from O₂ that we proposed for all hydrogenases embedded in redox-active films, including NiFe and standard FeFe hydrogenases, is also operational with CbA5H: A fraction of the incoming H₂ is sacrificed to reduce the incoming O₂ and prevent its penetration in the film thus protecting the enzyme within the film while a fraction of the enzyme near the film-electrolyte interface is inactivated (Fig. 4D and *Right* column of Fig. 3). However, with CbA5H, the inactivation of the enzyme that occurs near the film/solution interface is reversible. We have shown before, when we compared the immobilization of a NiFe hydrogenase and a more O₂-resistant variant of the same enzyme, that this reductive reactivation makes it possible to decrease the thickness of the film, thus increasing catalyst utilization without compromising long-term stability (40).

With the mediator in the right window of potential, the enzyme CbA5H, which is a very poor H₂ oxidation catalyst under conditions of DET, becomes very efficient in H₂ oxidation, revealing that the active site of CbA5H is not particularly tuned to H₂ evolution. The situation is reminiscent of two other FeFe hydrogenases from a different group (group B), the 3rd hydrogenase from *Clostridium pasteurianum* (CpIII) (45, 47) and that from
*Thermosediminibacter oceani* (ToHydA) (44), which inactivate under moderately oxidizing conditions as a result of the binding of a cysteine to the active site. The inactivation under oxidizing conditions makes these enzymes poor catalysts of H₂ oxidation in solution assays (45, 47). Our work with CbA5H shows that when this inactivation is prevented, the H₂ oxidation activity is recovered. It therefore appears that no FeFe hydrogenase identified so far is particularly tuned to be efficient in H₂ evolution, although various oxidative inactivation mechanisms may hide that they are efficient H₂ oxidation catalysts. This opens the perspective for a greater diversity of hydrogenases, beyond Cb5AH, which could benefit from integration into redox-active matrices to unlock their full potential for H₂ oxidation.

## Materials and Methods

The viologen-modified dendrimers (DV1 and DV2) and polymer (PV2) were synthesized and fully characterized according to previously reported procedures (32, 34).

Preparation of the [FeFe]-hydrogenase CbA5H from *C. beijerinckii* was carried out as previously described (20). For MET experiments, enzyme samples were exposed to air after in vitro maturation to induce $H_{inact}$ formation. The [FeFe]-hydrogenase DdHydAB from *D. desulfuricans* was prepared according to earlier work (48) and protected against inactivation by molecular oxygen through the addition of $Na_2S$ under oxidative conditions (25, 34, 48).

All electrochemical measurements were carried out in a three-electrode set-up consisting of a glassy-carbon rotating disc electrode (GC-RDEs, $d = 3$ mm, for MET) and a rotating pyrolytic graphite edge electrode (4 × 4 mm², for DET). A Pt-wire was used as a counter electrode and an Ag/AgCl 3 M KCl as a reference electrode. MET experiments were conducted in Tris/citrate buffer (50 mM) with KCl (100 mM) as the supporting electrolyte. DET experiments were conducted in phosphate buffer (100 mM) with NaCl (100 mM).

Enzyme films for DET experiments were prepared under anaerobic conditions by drop-casting 3 µL of a 10 µM solution of hydrogenase on a Pyrolytic Graphite Edge rotating disc electrode. The casted electrode was incubated for 3 min and rinsed with Milli-Q water to remove any unbound enzyme molecules.

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Unless stated otherwise, the hydrogel films for MET experiments were prepared by drop-casting a dendrimer-enzyme mixture of DV1/CbA5H (2.8 µg/0.16 nmol), DV2/CbA5H (0.57 µg/0.16 nmol), PV2/CbA5H (1.13 µg/0.16 nmol), or DV2/DdHydAB<sub>inact</sub> (0.28 µg/0.1 nmol) onto the GC-RDE. The films were allowed to dry for 2 h at 37 °C under an aerobic atmosphere. All CVs were recorded with an electrode rotating in a buffer predepegged with $N_2$ to create O₂-free conditions and then saturated with H₂. The DdHydAB<sub>inact</sub> in the hydrogel was reactivated via CV in H₂-saturated buffer.

To test the O₂-stability of CbA5H and DdHydAB in the absence of H₂ and under MET conditions, the enzymatic films were first activated via CVs under anaerobic conditions. Subsequently, the enzymatic films were exposed to oxygen (O₂) through storage under air at 5 °C. After exposure time, the residual activity was examined via CVs under anaerobic conditions. The procedure was repeated to determine the stability under repeated O₂ exposure. The O₂-stability of CbA5H during catalytic H₂ oxidation was investigated via CA with O₂ added at 5% into the H₂ gas feed.

**Data, Materials, and Software Availability.** Source data have been deposited in Zenodo (DOI: 10.5281/zenodo.15965654) (49). All other data are included in the article and/or SI Appendix.

**ACKNOWLEDGMENTS.** The financial support was provided by the Bundesministerium für Bildung und Forschung (BMBF) (Project ECO2DIS 031B1405D) to N.P., the European Innovation Council (Pathfinder project ECOMO, Grant No. 101115403), Horizon Europe (Innovation action CirculH2, Grant No. 101135542), L'Agence nationale de la recherche (ANR) and Deutsche Forschungsgemeinschaft (DFG) (PROSECCO, ANR-21-CE50-0041, PL 746/5-1), and the CNRS (IRP "Reve"). CL acknowledges funding by the French government under the France 2030 investment plan, as part of the Initiative d'Excellence d'Aix-Marseille Université - A*MIDEX, AMX-22-RE-AB-097. A*Midex is a foundation of Aix Marseille University. Jan Jaenecke acknowledges the financial support by "The German Academic Scholarship Foundation." D.T.F. acknowledges financial support by "Hans-Böckler-Stiftung". We thank Noura Shikh Issa for her help in drawing Fig. 1.

Author affiliations: <sup>a</sup>Technical University of Munich (TUM), Campus Straubing for Biotechnology and Sustainability, Uferstraße 53, Straubing 94315, Germany; and <sup>b</sup>CNRS, Aix-Marseille Universite, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille 13009, France

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