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The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S] ⇔ [4Fe-4S] and [Fe(I)Fe(II)] ⇔ [Fe(I)Fe(I)] thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H + 2e ⇄ H. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state H of the H-cluster actually consists of two species: H = [4Fe-4S] - [Fe(I)Fe(II)] and HH = [4Fe-4S] - [Fe(I)Fe(I)] (H) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S] subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (H/HH) has a pK ≈ 7.2.

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Hydrogenases are nature's efficient catalysts for both the generation of energy via oxidation of molecular hydrogen and the production of hydrogen via the reduction of protons. However, their O2 sensitivity and deactivation at high potential limit their applications in practical devices, such as fuel cells. Here, we show that the integration of an O2-sensitive hydrogenase into a specifically designed viologen-based redox polymer protects the enzyme from O2 damage and high-potential deactivation. Electron transfer between the polymer-bound viologen moieties controls the potential applied to the active site of the hydrogenase and thus insulates the enzyme from excessive oxidative stress. Under catalytic turnover, electrons provided from the hydrogen oxidation reaction induce viologen-catalysed O2 reduction at the polymer surface, thus providing self-activated protection from O2. The advantages of this tandem protection are demonstrated using a single-compartment biofuel cell based on an O2-sensitive hydrogenase and H2/O2 mixed feed under anode-limiting conditions.

Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. The most abundant hydrogenases contain a [NiFe] active site; these proteins are generally biased towards hydrogen oxidation activity and are reversibly inhibited by oxygen. However, there are [NiFe] hydrogenase that exhibit unique properties, including aerobic hydrogen oxidation and preferential hydrogen production activity; these proteins are highly relevant in the context of biotechnological devices. This review describes four classes of these "nonstandard" [NiFe] hydrogenases and discusses the electrochemical, spectroscopic, and structural studies that have been used to understand the mechanisms behind this exceptional behavior. A revised classification protocol is suggested in the conclusions, particularly with respect to the term "oxygen-tolerance". This article is part of a special issue entitled: metals in bioenergetics and biomimetics systems.

The orientation of hydrogenase bound covalently to a pyrolytic graphite edge electrode modified with a 4-aminophenyl monolayer can be modulated via electrostatic interactions during the immobilization step. At low ionic strength and when the amino groups of the electrode surface are mostly protonated, the hydrogenase is immobilized with the negatively charged region that surrounds its 4Fe4S cluster nearer to the protein surface facing the electrode. This allows direct electron transfer between the immobilized hydrogenase and the electrode, which is observed by the strong catalytic currents measured in the presence of the H2 substrate. Therefore, a very stable enzymatic electrode is produced that catalyzes nonmediated H2 oxidation.

Multiwalled carbon nanotubes grown on gold electrodes manufactured by microtechnology techniques have been used as a platform for oriented and stable immobilization of a Ni-Fe hydrogenase. Microscopic and electrochemical characterization of the system are presented. High-density currents due to H2 oxidation electrocatalysis, stable for over a month under continuous operational conditions, were measured. The functional properties of this nanostructured hydrogenase electrode are suitable for hydrogen biosensing and biofuel applications.

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation with high rates and efficiency under physiological conditions, but are highly oxygen sensitive. The [FeFe] hydrogenase from Desulfovibrio desulfuricans ( DdHydAB) can be purified under air in an oxygen stable inactive state H. The formation of the H state in vitro allows the handling of hydrogenases in air, making their implementation in biotechnological applications more feasible. Here, we report a simple and robust protocol for the formation of the H state in DdHydAB and the [FeFe] hydrogenase from Chlamydomonas reinhardtii, which is based on high potential inactivation in the presence of sulfide.