A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase.

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

Inhibition of [FeFe]-hydrogenases by formaldehyde and wider mechanistic implications for biohydrogen activation.

Formaldehyde-a rapid and reversible inhibitor of hydrogen evolution by [FeFe]-hydrogenases-binds with a strong potential dependence that is almost complementary to that of CO. Whereas exogenous CO binds tightly to the oxidized state known as H(ox) but very weakly to a state two electrons more reduced, formaldehyde interacts most strongly with the latter. Formaldehyde thus intercepts increasingly reduced states of the catalytic cycle, and density functional theory calculations support the proposal that it reacts with the H-cluster directly, most likely targeting an otherwise elusive and highly reactive Fe-hydrido (Fe-H) intermediate.

A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases.

In biology, rapid oxidation and evolution of H(2) is catalyzed by metalloenzymes known as hydrogenases. These enzymes have unusual active sites, consisting of iron complexed by carbonyl, cyanide, and thiolate ligands, often together with nickel, and are typically inhibited or irreversibly damaged by O(2). The Knallgas bacterium Ralstonia eutropha H16 (Re) uses H(2) as an energy source with O(2) as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an "O(2)-tolerant" hydrogenase. The mechanism of O(2) tolerance of Re MBH has been probed by measuring H(2) oxidation activity in the presence of O(2) over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O(2) and subsequent reactivation of the active site. Most significantly, O(2) tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H(2) affinity or the kinetics of O(2) attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O(2) tolerance in Re MBH that may be more widely applied to other [NiFe]-hydrogenases.

Formaldehyde--a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases.

Dihydrogen (H(2)) production by [FeFe]-hydrogenases is strongly inhibited by formaldehyde (methanal) in a reaction that is rapid, reversible, and specific to this type of hydrogenase. This discovery, using three [FeFe]-hydrogenases that are homologous about the active site but otherwise structurally distinct, was made by protein film electrochemistry, which measures the activity (as electrical current) of enzymes immobilized on an electrode; importantly, the inhibitor can be removed after addition. Formaldehyde causes rapid loss of proton reduction activity which is restored when the solution is exchanged. Inhibition is confirmed by conventional solution assays. The effect depends strongly on the direction of catalysis: inhibition of H(2) oxidation is much weaker than for H(2) production, and formaldehyde also protects against CO and O(2) inactivation. By contrast, inhibition of [NiFe]-hydrogenases is weak. The results strongly suggest that formaldehyde binds at, or close to, the active site of [FeFe]-hydrogenases at a site unique to this class of enzyme--highly conserved lysine and cysteine residues, the bridgehead atom of the dithiolate ligand, or the reduced Fe(d) that is the focal center of catalysis.

Hydrogen production under aerobic conditions by membrane-bound hydrogenases from Ralstonia species.

Studies have been carried out to establish the ability of O2-tolerant membrane-bound [NiFe] hydrogenases (MBH) from Ralstonia sp. to catalyze H2 production in addition to H2 oxidation. These hydrogenases are not noted for H2-evolution activity, and this is partly due to strong product inhibition. However, when adsorbed on a rotating disk graphite electrode the enzymes produce H2 efficiently, provided the H2 product is continuously removed by rapidly rotating the electrode and flowing N2 through the gastight electrochemical cell. Electrocatalytic H2 production proceeds with minimal overpotentiala significant observation because lowering the overpotential (the electrochemically responsive activation barrier) is seen as crucial in developing small-molecule catalysts for H2 production. A mutant having a high KM for H2 oxidation did not prove to be a better H2 producer relative to the wild type, thus suggesting that weak binding of H2 does not itself confer a tendency to be a H2 producer. Inhibition by H2 is much stronger than inhibition by CO and, most significantly, even O2. Consequently, H2 can be produced sustainably in the presence of O2 as long as the H2 is removed continuously, thereby proving the feasibility for biological H2 production in air.

Dynamic electrochemical investigations of hydrogen oxidation and production by enzymes and implications for future technology.

This tutorial review describes studies of hydrogen production and oxidation by biological catalysts--metalloenzymes known as hydrogenases--attached to electrodes. It explains how the electrocatalytic properties of hydrogenases are studied using specialised electrochemical techniques and how the data are interpreted to allow assessments of catalytic rates and performance under different conditions, including the presence of O2, CO and H2S. It concludes by drawing some comparisons between the enzyme active sites and platinum catalysts and describing some novel proof-of-concept applications that demonstrate the high activities and selectivities of these 'alternative' catalysts for promoting H2 as a fuel.