Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough under Oxidative Conditions

We report on the fabrication of bioanodes for H2 oxidation based on [NiFeSe] hydrogenase. The enzyme was electrically wired by means of a specifically designed low-potential viologen-modified polymer, which delivers benchmark H2 oxidizing currents even under deactivating conditions owing to efficient protection against O2 combined with a viologen-induced reactivation of the O2 inhibited enzyme. Moreover, the viologen-modified polymer allows for electrochemical co-deposition of polymer and biocatalyst and, by this, for control of the film thickness. Protection and reactivation of the enzyme was demonstrated in thick and thin reaction layers.

H ydrogenases bearing the [NiFeSe] active site are of particular interest for energy conversion because of their ability to produce H 2 in the presence of certain amounts of O 2 . 1−7 This behavior was attributed to their unique structural and chemical properties, which are related to the selenocysteine ligand coordinating the Ni−Fe active site. 7−10 Under rather mild reducing conditions (≈ < 0 V vs SHE) and in a direct electron transfer (DET) regime between an adsorbed enzyme and electrode surface, the inactive enzyme is reduced and full activity is restored. 2,11,12 Consequently, [NiFeSe] hydrogenases show very fast rates of reactivation in the presence of O 2 under reducing conditions (H 2 production). Hence, these types of hydrogenases are very promising catalysts for H 2 evolution, and they have been successfully employed as active material in photocatalytic hydrogen production devices. [3][4][5][6]13 On the other hand, the fast reactivation rate at negative potentials is accompanied by fast deactivation under both oxidizing electrochemical and aerobic conditions. 2,9,11 Hence, the application of [NiFeSe] hydrogenases as biocatalysts in bioanodes was considered unpractical, 2 despite the fact that [NiFeSe] hydrogenases have among the highest activities toward the oxidation of H 2 (up to 4000 s −1 for [NiFeSe] from Desulfomicrobium baculatum). 2 We demonstrated recently that fragile catalysts such as highly O 2 -sensitive [NiFe] or [FeFe] hydrogenases can be protected from inactivation by O 2 by their incorporation into an oxygenreducing redox matrix, for example, a viologen-modified redox hydrogel. 14−16 Electrons that are generated by the conversion of H 2 into protons are transferred to the polymer-bound viologen moieties and are used to reduce incoming O 2 at the polymer−electrolyte interface. 14−16 The reduced viologens were also proposed to reactivate O 2 deactivated NiFe hydrogenase when a considerable amount of H 2 was present. 17 Moreover, viologen-based polymers act as a Nernst buffer and protect the hydrogenases from high potential deactivation. 14 Stimulated by these results, we use the highly active [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough (DvH-[NiFeSe], recombinant form, H 2 uptake activity: 2400 s −1 ) 18 to set a new benchmark for H 2 oxidation currents with a fragile enzyme in a stabilizing redox polymer matrix. We demonstrate that the concept based on thick redox polymer films with O 2 blocking properties 14−16 is transposable to the efficient protection of [NiFeSe] hydrogenase. More importantly, by exploiting specifically designed thin polymer/ enzyme films, we reveal the previously unknown reactivation capability through the redox matrix, which is essential for catalysts displaying fast oxidative deactivation rates such as the [NiFeSe] hydrogenase.
We implemented a novel viologen-modified polymer, which allows for the formation of enzyme/polymer films by means of a standard drop cast (thick f ilms) and of an electrochemically induced deposition process (thin f ilms). The latter is based on the in situ deprotection of protected bifunctional crosslinkers. 19 For this, the polymer backbone P(N 3 MA-BA-GMA) was synthesized in a multistep synthesis strategy starting from the monomers N 3 MA, GMA, and BA in a nominal ratio of 80:10:10, respectively (Scheme 1; for synthesis of the N 3 MA monomer, see the SI). Analysis of the integrals in the 1 H NMR spectrum of the polymer backbone ( Figure S1a) revealed an actual composition of 71:20:9, which slightly deviates from the nominal composition derived from the monomer ratio. The polymer backbone bears two functionalities, that is, N 3 (Scheme 1a, blue) and epoxide (red) groups, which allow for covalent attachment of an alkyne-modified viologen derivative and reaction with bifunctional cross-linkers, respectively.
The alkyne-modified viologen-based redox mediator vio (Scheme 1; for synthesis, see the SI) was attached to the polymer backbone P(N 3 MA-BA-GMA) in a Cu(I)-catalyzed 1,3-dipolar cycloaddition ("click" chemistry) to yield the target redox polymer P(N 3 MA-BA-GMA)-vio. NMR and IR characterization of P(N 3 MA-BA-GMA)-vio ( Figure S1b Figure S2). The potential of the first reduction is above the H 2 /2H + couple (≈−450 mV at pH 7). Hence, the polymer-tethered viologen should be able to accept electrons from the polymer-integrated hydrogenase. Moreover, this value is more negative than the reactivation potentials of the [NiFeSe] hydrogenase. From a

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Letter thermodynamic point of view, the viologen should be hence able to reduce the inactivated hydrogenase. It should be noted that, compared to the freely diffusing mediator vio, the potentials of both reductions (−451 and −683 mV, Figure  S1, blue line) are shifted to more positive potentials in the case of the polymer-bound viologen unit (−295 and −660 mV, Figure S1, black line). Thus, the polymer matrix stabilizes the reduced forms of the mediator. Figure 1a depicts cyclic voltammograms of a P(N 3 MA-BA-GMA)-vio/DvH-[NiFeSe] film drop cast onto a glassy carbon (GC) electrode under argon (black line) and H 2 atmospheres (red lines). Under an argon atmosphere, a chemically reversible redox couple of the first viologen reduction was observed (black curve). Under turnover conditions (red line, 100% H 2 ), a pronounced catalytic current response was detected, with a half-wave potential that closely matches the redox potential of the viologen moiety. Because the latter is ∼150 mV more positive than the H 2 /2H + couple at pH 7.4, the enzyme is productively wired via the polymer-tethered viologen moieties and DET can be excluded. Moreover, steady-state currents were observed for high (100%, Figure 1a, red solid line) and low (5%, red dashed line) H 2 concentrations at potentials between ∼−0.2 and +0.2 V even for slow scan rates (2 mV s −1 , Figure S3a). We conclude that the redox polymer acts as a Nernst buffer and that high potential deactivation, which starts between −0.1 V and 0 V at low H 2 concentrations, 2,11 is absent. No significant loss of catalytic current was observed within a period of 16 h under continuous turnover conditions ( Figure  S3b), and the peak currents of the viologen moiety show almost identical intensity before and after the long-term experiment ( Figure S3c). Moreover, electrodes that were prepared from the same enzyme batch show a similar catalytic response ( Figure  S4). Figure 1b exhibits an I−t curve of a drop-cast P(N 3 MA-BA-GMA)-vio/DvH-[NiFeSe] film measured at an applied potential of +160 mV (vs SHE) with 20% H 2 /80% Ar bubbling through the electrolyte. At this H 2 partial pressure, the amount of H 2 in solution defines the catalytic current ( Figure S5a), which is attributed to H 2 mass transport limitations within thick films. 16 Upon addition of 5% O 2 to the gas feed (5% O 2 /20% H 2 /75% Ar), the current decreases, indicating that electrons from the enzyme-catalyzed oxidation of H 2 are used by the viologen species to reduce incoming O 2 . 14, 16 Note that at this high potential O 2 is not reduced at the GC electrode ( Figure  S5b) but exclusively via the reduced viologen species. Alternating between the H 2 /O 2 /Ar and the H 2 /Ar gas feeds reveals that the catalytic current for H 2 oxidation recovers during each anaerobic period (Figure 1b). In contrast, complete loss of the catalytic current is observed for enzymes wired in the DET regime.  hydrogenases; a so-called high potential reactivating species 2 or "fast" inactive state 11 as well as a low potential reactivating species 2 or "slow" inactive state, 11 which can be reactivated at potentials around ∼0 and ∼−0.35 V (vs SHE), respectively. Both are above or close to the redox potential of the polymerbound viologen unit (∼−0.3 V).
Deactivation by O 2 and reactivation processes of an enzyme embedded in a redox matrix are best studied in thin polymer/ enzyme layers to limit the contribution from protection through O 2 reduction (which increases with the film thickness 16 ). However, the formation of very thin films by a common drop-cast process is difficult to achieve in a reproducible way. Hence, we applied a nonmanual deposition process based on electrochemical in situ activation of a protected bifunctional cross-linker, that is, trityl-protected 2,2′-(ethylenedioxy)-bis(ethylamine) (CL p , Scheme 1b). Deprotection occurs by applying short positive potential pulses (+1.71 V vs SHE for 0.2 s), which lead to water splitting at the electrode surface and thus a change in the local pH value within the diffusion layer by generation of H + ions. Under these conditions, the pH-responsive trityl protecting groups are cleaved from the amino groups of the cross-linker (Scheme 1b). The activated cross-linker (Cl a ) starts then to react with the electrophilic epoxide functions within P(N 3 MA-BA-GMA)-vio (Scheme 1b) and forms a dense, insoluble 3D network that is able to entrap the biocatalyst. The amount and hence the film thickness of the deposited polymer can be adjusted by varying the number n of repetitions of the applied potential pulse sequence. Between pulses, a potential of +0.21 V vs SHE was applied for 2 s to allow the reaction to proceed and to refresh the cross-linker and polymer concentration at the electrode surface. This led to a pulse sequence of n (+1.71 V/0.2 s; +0.21 V/2 s). Preliminary tests with the pure polymer revealed that the maximum loading of polymer with respect to the amount of viologen species on the electrode was obtained for n = 20−30 ( Figure S6 and Table S1, the highest peak current and highest specific viologen surface concentration). Optical micrographs of the deposited films showed the deposition of many small polymer spots on the electrode surface rather than a homogeneous film ( Figure S7). However, the amount of deposited material is substantially lower than that in the case of a drop-cast film. A closer inspection of the cyclic voltammograms revealed that only for n < 20 were very thin films that exhibit characteristics of an adsorption-controlled electron transfer reaction obtained (peak current ∝ scan rate, peak potential separation < 60 mV; see, e.g., Figure S6c,e). 100% argon (black line) and 100% H 2 (red solid line). By calculating the charge from voltammograms recorded at a scan rate of 2 mV s −1 under nonturnover conditions, the surface concentration of viologen moieties Γ vio was estimated to be 3.32 nmol cm −2 , which is ∼40 times lower than that for a dropcast polymer film ( Figure S2). A pronounced catalytic current response with a steady-state current above ∼−0.2 V was observed under turnover conditions, even for low H 2 concentrations (5% O 2 , red dashed line) and for scan rates of 10 mV s −1 , demonstrating that the viologen-based redox matrix acts as a Nernst buffer even in thin films, which is in contrast to the DET regime, where high potential deactivation was observed at this scan rate. 2 The absolute catalytic currents correlate with n ( Figure S8a), and even for small values (n = 1 or 2), pronounced catalytic currents were obtained ( Figure  S8b,c). Moreover, the fact that the catalytic current correlates with the number of applied deposition cycles n ( Figure S8a) clearly demonstrates that the observed catalytic currents are limited by the amount of biocatalyst (for n < 10) on the electrode surface and mass transport is not the rate-limiting step. 20,21 Chronoamperometry under 20% H 2 /80% Ar (Figure 2b, black trace) of a very thin film deposited with n = 2 revealed that after adding O 2 to the gas feed (t = 550 s, 5% O 2 /20% H 2 / 75% Ar) the enzyme within the film was fully deactivated within ∼300 s. In contrast to drop-cast films, no current was recovered when the O 2 feed was stopped (t = 900 s, 20% H 2 / 80% Ar), indicating that all enzyme molecules were deactivated. However, when a negative potential was applied to the electrode (−390 mV vs SHE, at t = 1200 to 1500 s) with 20% H 2 /80% Ar bubbling through the cell, the oxidative current could be fully restored (at t > 1500 s). Obviously, the use of low-potential viologen moieties allows for reactivation of the enzymes, in analogy with observations in the DET regime. Because a DET between the electrode and the enzyme can be excluded for our system (no deactivation at high potentials), reactivation of the enzyme proceeds via the reduction of the inactive enzyme by the reduced viologen species. This was evidenced by experiments where potentials above the redox potential of the viologen moiety were applied after the O 2 flow was stopped (−0.1 and 0 V vs SHE, Figure S9a,b). In this case, reactivation was not observed for potentials of −0.1 V ( Figure  S9a) or 0 V ( Figure S9b). Only when the potential was stepped to values more negative than the redox potential of the viologen (∼−0.3 V) could catalytic currents be restored (−0.39 V; Figure S9c).

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It should be noted that no conclusion on the type and nature of the inactive state of the hydrogenases can be drawn because the potential of the viologen matrix (−0.295 V) is below or very close to the reactivation potentials of the low (∼−0.350 V) and high potential (∼0 V) reactivating species. 2,11 The degree of current decay upon addition of O 2 clearly depends on the number of n used for polymer deposition and thus on the film thickness (Figures 2b and S10a−c). Films that were deposited with n = 10 ( Figure 2b, blue trace) do not show complete deactivation upon addition of O 2 possibly due to increasing contribution from protection through O 2 reduction. This is backed by the fact that when the O 2 flux was stopped thick films showed at least partial recovery of the current ( Figure S10e). Moreover, this demonstrates that deactivation is indeed based on inhibition by O 2 and not due to high potential deactivation that would occur in the DET regime. Reactivation is also possible under a pure argon atmosphere ( Figure S10d,e). We conclude that for the viologen-induced reactivation process, the presence of H 2 is not a prerequisite.
In conclusion, the use of a low potential redox polymer ensures efficient protection of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough against O 2 and thus enables application of this particular enzyme as a catalyst for H 2 oxidation. Current densities of 1.0 and 1.7 mA cm −2 , obtained for thin (favorable mass transport) and thick (high catalyst loading) polymer/enzyme films, respectively, are the highest for hydrogenase incorporated in redox matrices. The low potential redox mediator does not only allow for protection through reduction of molecular oxygen but also enables reactivation of deactivated biocatalyst by transferring electrons to the inhibited enzyme, as was shown in thin films, deposited via an electrochemically induced cross-linking process. Our results clearly demonstrate that the combination of this particular hydrogenase and viologen-based redox hydrogels is