Photocatalytic Hydrogen Evolution with a Hydrogenase in a Mediator-Free System under High Levels of Oxygen

The development of solar water-splitting systems provides a route to renewable H₂.[1] A prerequisite for an efficient water-splitting process is the availability of highly efficient, inexpensive, and stable catalysts for H₂ and O₂ production. Despite much progress by synthetic and materials chemists,[2] it is still the natural enzymes that set the benchmark efficiency in the reduction and oxidation of water.[3] [FeFe] and [NiFe] hydrogenases convert protons and electrons into H₂ with remarkably high rates at a low overpotential.[4] Photocatalytic H₂ generation has previously been reported with hydrogenases in a heterogeneous scheme with the enzyme attached directly to semiconducting particles or electrodes.[5] Homogeneous systems with a dye and hydrogenase in solution are well established, but these multicomponent systems require a soluble redox mediator to transport the electron from the light absorber to the catalyst.[6]

An important requirement for water splitting is that the catalyst for H₂ evolution tolerates at least small levels of O₂, which will enter the system either through leakage of atmospheric O₂ into the photoreactor and/or from the in situ formation of O₂ during the water-splitting process.[7] Although many hydrogenases, in particular [FeFe] hydrogenase, are highly sensitive to O₂, much progress has recently been reported in identifying and understanding the factors leading to O₂ tolerance in [NiFe] hydrogenases.[8] In addition, a photocatalytic H₂ evolution system without a redox mediator is desirable, because a chemically reduced mediator is easily quenched by O₂, which can result in drastically reduced photoactivity for the system in the presence of O₂.

Herein we report on a photocatalytic H₂ evolution system consisting of a Desulfomicrobium baculatum (Db) [NiFeSe] hydrogenase and an organic dye, Eosin Y (EY), which evolves H₂ photocatalytically under high levels of O₂ (Figure 1). Db [NiFeSe] hydrogenase was selected as the H₂ evolution catalyst because it displays unique properties among hydrogenases.[9] This enzyme has been reported to be biased towards H₂ evolution, showing electrocatalytic H₂ production activity in the presence of as much as 1 % O₂ and displaying little product inhibition.[10] This hydrogenase has previously been adsorbed on ruthenium-dye-sensitized TiO₂ nanoparticles for the photocatalytic generation of H₂, but the formation of reactive oxygen species (ROS) on TiO₂ during irradiation prevented its use in the presence of O₂.[5a, 11] In this study, we removed the necessity for TiO₂ by replacing dye-sensitized TiO₂ with soluble EY, which allows for photo-induced direct electron transfer to a catalyst.[7,12] Elimination of radical-forming TiO₂ allowed us to produce H₂ photocatalytically in our hydrogenase-based system under remarkably high levels of O₂.

Figure 1.

Schematic representation of photocatalytic H₂ generation with EY and an O₂-tolerant Db [NiFeSe] hydrogenase (EY–hydrogenase) system in the presence of TEOA in pH-neutral aqueous solution. Photo-induced electron transfer occurs directly from the EY to the [Fe₄S₄] cluster relay[3a, 6c, 8g] and then to the H₂-evolving [NiFeSe]-active site of the enzyme (see text).

First, the photocatalytic activity was studied of a homogeneous aqueous solution of Db [NiFeSe] hydrogenase and EY in the presence of the electron donor triethanolamine (TEOA) under an inert atmosphere. Optimized conditions were obtained by varying the amount of hydrogenase, EY, and TEOA and the pH of the solution (Figures S1–S3). The EY–hydrogenase system worked efficiently in the absence of any soluble redox mediator when a stirred solution of hydrogenase (10 pmol) and EY (disodium salt, 1 μmol) in TEOA (2.25 mL, 150 mm) at pH 7 and 25 °C was exposed to visible light (solar light simulator; AM 1.5 G, 100 mW cm⁻², λ>420 nm). The photoreactor was purged prior to the experiment with N₂ containing 2 % CH₄ (internal standard for gas chromatography (GC) measurements, see the Supporting Information).

The EY–hydrogenase system photogenerated (0.50±0.03) μmol of H₂ per hour and almost linear H₂ evolution rates up to 15 hours (Figure S4). This result corresponds to a hydrogenase-based turnover frequency (TOF_hydrogenase) of (13.9±0.7) (mol H₂)(mol hydrogenase)⁻¹ s⁻¹. The photoactivity of the system is lost after 24 h, whereupon (5.0±0.3) μmol of H₂ had accumulated, corresponding to a TON_hydrogenase of (5.0±0.3)×10⁵ and an EY-based TON (TON_EY) of (5.0±0.3) (mol H₂)(mol EY)⁻¹. No H₂ was detected in the dark or in the absence of EY or hydrogenase. At 5 °C and 45 °C TOF_hydrogenase values of (7.5±0.5) s⁻¹ and (19±2) s⁻¹ were obtained, respectively. The system exhibits an optimum activity at pH 7.0 and its performance is decreased by more than 50 % at pH 6.0 and 8.0 (Figure S2). This can be explained by the lower intrinsic activity of the hydrogenase under basic conditions[10] and an increased amount of protonated TEOA donor under an acidic environment.[5a, 12a] The EY–hydrogenase system operates with high photoactivity in the absence of any soluble redox mediator and electrons are transfered directly from the dye to the hydrogenase. The per-active-site performance of the EY–hydrogenase system is on the same order of magnitude as that of a previously reported system with Db [NiFeSe] hydrogenase on ruthenium-dye-sensitized TiO₂ (TOF_hydrogenase up to 50 s⁻¹)[5a] and much higher than a photocatalytic system with EY and a synthetic Co catalyst (TOF_Co=0.02 s⁻¹)[7] in an aqueous pH-neutral TEOA solution.

Variation of the light intensity of monochromatic LED light (525 nm; pH 7.0 and 25 °C) from 1.5 to 5 and finally 18 mW cm⁻² resulted in external quantum efficiencies (EQE) of (1.50±0.08), (0.49±0.03), and (0.18±0.01) % with a corresponding TOF_hydrogenase of (16±1), (18±1), and (24±1) (mol H₂)(mol hydrogenase)⁻¹ s⁻¹, respectively. The EQE increases with decreasing light intensity, whereas the TOF_hydrogenase changes only marginally. The TOF_hydrogenase also remained almost constant when the light intensity of visible light was increased from 50 to 100 mW cm⁻² (Figure S5). At 100 mW cm⁻² visible-light irradiation, an increasing amount of EY from 1 to 3 μmol did not result in the photogeneration of higher amounts of H₂. Furthermore, when the amount of hydrogenase was increased from 10 to 50 pmol the amount of H₂ photogenerated in the system more than doubled (Table S1, Figures S1 and S3). These experiments demonstrate that the hydrogenase limits the EY–hydrogenase system, which is an important requirement for studying the effect of inhibitors on the enzyme in the photocatalytic system.

Subsequently, the photocatalytic H₂ production activity of the EY–hydrogenase system was investigated in the presence of varying concentrations of O₂. Previously, protein film electrochemistry with Db [NiFeSe] hydrogenase on a pyrolytic graphite edge electrode demonstrated that this enzyme evolves H₂ in the presence of 1 % O₂ at an applied potential of −0.45 V versus the normal hydrogen electrode (NHE) in an aqueous electrolyte solution at pH 6.0.[10] Thus, H₂ evolution under O₂ should be possible if the photoexcited EY dye can efficiently transfer electrons directly to the hydrogenase.

To test this hypothesis, the EY–hydrogenase system (10 pmol of hydrogenase and 1 μmol of EY in 2.25 mL of aqueous 150 mm TEOA solution at pH 7 and 25 °C) was irradiated for one hour under N₂ atmosphere (with 2 % CH₄) to verify its activity under inert atmosphere. The photo-reactor was purged with 2 % CH₄/N₂ and different amounts of O₂ were injected into the headspace after 1 hour with subsequent irradiation (Table 1 and Figure 2). In all cases, the photoactivity of the EY–hydrogenase system decreased with an increasing O₂ concentration in the headspace. Remarkably, in the presence of 21 % O₂ some photoactivity still remained ((11±3) % of the photoactivity under anaerobic conditions; Figure 2, insert).

Table 1.

Visible-light-driven (100 mW cm⁻², AM 1.5 G, λ>420 nm) H₂ production with EY–hydrogenase in an aqueous TEOA solution (2.25 mL, 150 mm) at pH 7 and 25 °C.

Conditions	TOFhydrogenase[a] [s−1]	H2[a] [μmol H2 h−1]
EY (1 μmol) and hydrogenase (10 pmol)
0 % O2	13.9±0.7	0.50±0.03
5 % O2	11.5±1.0	0.41±0.04
10 % O2	7.3±0.4	0.26±0.01
15 % O2	4.5±0.5	0.16±0.02
21 % O2	1.5±0.3	0.05±0.01
2 % CO	<0.4	<0.02
[Ru(bipy)3]2+ (1 μmol), hydrogenase (10 pmol), and MV (1 μmol)
0 % O2	27±2	0.98±0.08
5 % O2	3.3±0.5	0.12±0.02

^[a]Calculated based on the amount of H₂ produced in the first 0.5 h of irradiation; standard deviation for at least three experiments.

Figure 2.

Amount of H₂ generated with the EY–hydrogenase system in an aqueous TEOA solution (150 mm, pH 7.0) during visible-light irradiation (t_irr, 100 mW cm⁻², AM 1.5 G, λ>420 nm) at 25 °C. The EY–hydrogenase system was exposed to different O₂ headspace concentrations after 1 h of irradiation under 2 % CH₄/N₂. Insert: Relative photocatalytic H₂ evolution activity compared to that under anaerobic conditions (based on photoactivity within the first 0.5 h of irradiation).

The concentration of O₂ was also measured in the solution for experiments with various concentrations of O₂ in the headspace (Table S2). Irradiation of the EY–hydrogenase system under an atmosphere of 21 % O₂ resulted in decrease of the concentration of dissolved O₂ from (6.0±1.0) ppm to below 0.2 ppm within 1 min of irradiation, thereby creating conditions conducive for the O₂-tolerant hydrogenase. The same behavior was observed in the absence of hydrogenase and can be ascribed to the known reaction of photo-excited EY with O₂, resulting in the formation of singlet O₂.[13] EY was used in excess in the system and the quenching of photo-excited EY with O₂ explains the rapid decrease in the amount of dissolved O₂ and the slow depletion of headspace O₂ after several hours (Figure S6). Irradiation of the solution under a high concentration of O₂ in the headspace presumably also resulted in the formation of increased amounts of singlet O₂, causing the decreased lifetime of the photo-H₂ evolution system (Figure 2).

The EY–hydrogenase system is fully photoactive after three hours under anaerobic conditions, but it is inactivated after three hours of irradiation under a 21 % O₂ atmosphere. This endurance is remarkable when one considers the complete photo-decomposition of the same hydrogenase under air on dye-sensitized TiO₂ within 2 min, which is presumably due to the decomposition of the enzyme by ROS formed upon reduction of O₂ by conduction band electrons.[5a, 11] Notable differences between the dye–TiO₂–hydrogenase and EY–hydrogenase systems are the generation of radical species in close proximity to the hydrogenase in the former case, whereas singlet O₂ is mainly produced remote from the enzyme in the latter system.

In related work, a photocatalytic H₂ evolution system consisting of a ruthenium dye covalently linked to [NiFe] hydrogenase from Thiocapsa roseopersicina in the presence of the soluble redox mediator methyl viologen (MV) was exposed to O₂ in a closed photoreactor.[6b] Initial irradiation did not show formation of H₂, but resulted in the depletion of O₂ in the system, whereupon an anoxic environment in the system allowed for reactivation of the enzyme and formation of H₂.[6b]

Therefore, we also tested a homogeneous system comprising [Ru(2,2′-bipyridine)₃]Cl₂ (1 μmol), [NiFeSe] hydrogenase (10 pmol), and MV (1 μmol) for comparison with the EY–hydrogenase system. The multicomponent Ru–MV–hydrogenase system is only photo-active in the presence of MV because electron transfer does not occur from photoexcited Ru directly to the hydrogenase.[5a, 6a,b] Under anaerobic conditions, the Ru–MV–hydrogenase system evolves (0.98±0.08) μmol H₂ h⁻¹ with a TOF_hydrogenase of (27±2) mol H₂ (mol hydrogenase)⁻¹ s⁻¹ during visible-light irradiation. In the presence of 5 % headspace O₂, the photoactivity decreased dramatically to (3.3±0.5) s⁻¹ and (0.12±0.02) μmol H₂ h⁻¹ (Table 1) and no significant amounts of H₂ were observed under 21 % O₂. Photoexcited [Ru(2,2′-bipyridine)₃]²⁺ and reduced MV react with O₂,[14] resulting in almost complete inactivation of the system.

In order to investigate the reversibility of the inhibitory effect of O₂ in the EY–hydrogenase system, reactivation of hydrogenase under inert conditions after exposure to atmospheric O₂ (21 %) was also examined. The EY–hydrogenase system was exposed to aerobic conditions for different periods of time at continuous white-light irradiation. After repurging the photoreactor with 2 % CH₄/N₂, we measured the photocatalytic H₂ production. The resulting TOF dropped to (81±4), (65±6), and (31±3) % of the initial value after 30, 60, and 120 min of continuous exposure to air and light, respectively (Table S3 and Figure S7). Control experiments with EY–hydrogenase exposed to air in the dark for the same duration has led to a negligible inactivation of the hydrogenase (Table S3 and Figure S7).

Thus, the hydrogenase displays a good robustness in the presence of air and can be partially reactivated under inert conditions following light exposure with EY. Exposure of [NiFeSe] hydrogenases to O₂ results in the oxidation of sulfur and/or selenium in cysteine and selenocysteine ligands at the active site.[9b–d] These inactive states can be reactivated through reduction and recovery of the amino acids. Exposing the EY–hydrogenase system to light results in photoexcitation of EY and formation of a long-lived triplet state.[12] Low-potential electrons are transferred to the hydrogenase, thereby reactivating O₂-inactive states in the hydrogenase.[10] This fast reactivation of any O₂-inactivated hydrogenase can be explained by the negative excited state reduction potential of EY (*EY=−0.91 V vs. NHE).[12a]

The effect of carbon monoxide, a well-known inhibitor of hydrogenases,[10] on the photocatalytic formation of H₂ was also tested with EY–hydrogenase. Introduction of 2 % CO in N₂ resulted in complete inactivation of the EY–hydrogenase system (Figure S8) and only a negligible amount of H₂ was detectable by GC. Inhibition by CO was at least partially reversible and purging the inactive system with N₂ containing 2 % CH₄ resulted in (52±3) % of the initial TOF_hydrogenase activity. The reversibility of CO inhibition is in agreement with the previously reported electrochemical studies.[10]

In conclusion, a photocatalytic H₂ evolution system with an O₂-tolerant Db [NiFeSe] hydrogenase and EY was assembled, which contains solely earth-abundant materials and maintains photoactivity under remarkably high levels of headspace O₂. H₂ evolution is driven efficiently by photoinduced direct electron transfer from EY to hydrogenase, making a soluble redox mediator unnecessary and thereby allowing for remarkable photostability under high O₂ levels. This work demonstrates an unprecedented robustness of a hydrogenase towards O₂ and paves the way to the exploitation of hydrogenases in full water splitting.

Supporting Information

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