In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]H subcluster, a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]H subcluster and one iron of the cubane subcluster. The other iron in the [2Fe]H subcluster is called Fe2. The light sensitivity of the Desulfovibrio desulfuricans enzyme in a variety of states has been studied with infrared (IR) spectroscopy. The aerobic inactive enzyme (H(inact) state) and the CO-inhibited active form (H(ox)-CO state) were stable in light. Illumination of the H(ox) state led to a kind of cannibalization; in some enzyme molecules the H cluster was destroyed and the released CO was captured by the H clusters in other molecules to form the light-stable H(ox)-CO state. Illumination of active enzyme under 13CO resulted in the complete exchange of the two intrinsic COs bound to Fe2. At cryogenic temperatures, light induced the photodissociation of the extrinsic CO and the bridging CO of the enzyme in the H(ox)-CO state. Electrochemical redox titrations showed that the enzyme in the H(inact) state converts to the transition state (H(trans)) in a reversible one-electron redox step (E (m, pH 7) = -75 mV). IR spectra demonstrate that the added redox equivalent not only affects the [4Fe-4S]H subcluster, but also the di-iron centre. Enzyme in the H(trans) state reacts with extrinsic CO, which binds to Fe2. The H(trans) state converts irreversibly into the H(ox) state in a redox-dependent reaction most likely involving two electrons (E (m, pH 7) = -261 mV). These electrons do not end up on any of the six Fe atoms of the H cluster; the possible destiny of the two redox equivalents is discussed. An additional reversible one-electron redox reaction leads to the H(red) state (E (m, pH 7) = -354 mV), where both Fe atoms of the [2Fe]H subcluster have the same formal oxidation state. The possible oxidation states of Fe1 and Fe2 in the various enzyme states are discussed. Low redox potentials (below -500 mV) lead to destruction of the [2Fe]H subcluster.

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A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H+/H2 redox couple, and H2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O2 vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O2, surviving only if exposed to O2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O2 even during turnover and continues to catalyze H2 oxidation in the presence of O2.

Redox enzymes can be adsorbed onto electrode surfaces such that there is a rapid and efficient direct electron transfer (ET) between the electrode and the enzyme's active site, along with high catalytic activity. In an idealized way, this may be analogous to protein-protein ET or, more significantly, the nonrigid interface between different domains of membrane-bound enzymes. The catalytic current that is obtained when substrate is added to the solution is directly proportional to the enzyme's turnover rate and its dependence on the electrode potential reports on the energetics and kinetics of the entire catalytic cycle. Although the current is expected to reach a limiting value as the electrode potential is varied to increase the driving force, a residual slope in voltammograms is often observed. This slope is significant, as it is unexpected from all simple considerations of electrochemical kinetics. A particularly remarkable result is obtained in experiments carried out with the [NiFe]-hydrogenase from Allochromatium Vinosum: this enzyme displays high catalytic activity for hydrogen oxidation and is easily studied up to 60 °C, at which temperature the current-potential response becomes completely linear over a range of more than 0.5 V. The explanation for this effect is that the enzyme molecules are not adsorbed in a homogeneous manner but vary in their degree of ET coupling with the electrode, i.e., through there being many slightly different orientations. Under conditions in which interfacial ET becomes rate-limiting, i.e., when turnover number is high at elevated temperatures, the current-potential response reflects the superposition of numerous electrochemical rate constants. This is highly relevant in the interpretation of the catalytic electrochemistry of enzymes.

The cycling between active and inactive states of the catalytic center of [NiFe]-hydrogenase from Allochromatium vinosum has been investigated by dynamic electrochemical techniques. Adsorbed on a rotating disk pyrolytic graphite "edge" electrode, the enzyme is highly electroactive: this allows precise manipulations of the complex redox chemistry and facilitates quantitative measurements of the interconversions between active catalytic states and the inactive oxidized form Ni(r) (also called Ni-B or "ready") as functions of pH, H(2) partial pressure, temperature, and electrode potential. Cyclic voltammograms for catalytic H(2) oxidation (current is directly related to turnover rate) are highly asymmetric (except at pH > 8 and high temperature) due to inactivation being much slower than activation. Controlled potential-step experiments show that the rate of oxidative inactivation increases at high pH but is independent of potential, whereas the rate of reductive activation increases as the potential becomes more negative. Indeed, at 45 degrees C, activation takes just a few seconds at -288 mV. The cyclic asymmetry arises because interconversion is a two-stage reaction, as expected if the reduced inactive Ni(r)-S state is an intermediate. The rate of inactivation depends on a chemical process (rearrangement and uptake of a ligand) that is independent of potential, but sensitive to pH, while activation is driven by an electron-transfer process, Ni(III) to Ni(II), that responds directly to the driving force. The potentials at which fast activation occurs under different conditions have been analyzed to yield the potential-pH dependence and the corresponding entropies and enthalpies. The reduced (active) enzyme shows a pK of 7.6; thus, when a one-electron process is assumed, reductive activation at pH < 7 involves a net uptake of one proton (or release of one hydroxide), whereas, at pH > 8, there is no net exchange of protons with solvent. Activation is favored by a large positive entropy, consistent with the release of a ligand and/or relaxation of the structure around the active site.

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.

In this report the first high-quality infrared spectra of [Fe]-hydrogenase are presented. Analyses of these spectra obtained under a variety of redox conditions strongly indicate that [Fe]-hydrogenases contain a low-spin Fe ion in the active site with one CN Ϫ group and one CO molecule as intrinsic, non-protein ligands. When in the ferric state, the presence of such an ion can explain the enigmatic EPR properties (the rhombic 2.10 signal) of the active, oxidised enzyme. To account for other, well-characterised properties of the active site, we propose that the active site of [Fe] A third class of hydrogenase, not containing any metals and only active in the presence of its cofactor, has been discovered in methanogenic Archaea by Thauer and coworkers [8,9].A few [NiFe]-hydrogenases have been extensively studied. The basic unit of these enzymes is formed by a large (47Ϫ 72 kDa) and a small (23Ϫ38 kDa) subunit. In the last 4 years FTIR studies on the enzymes from Chromatium vinosum [10Ϫ 13] and Desulfovibrio gigas [14,15], combined with the crystal structure of the D. gigas enzyme [14,16] have revealed that the active site is a bimetallic NiFe site with two CN Ϫ groups and one CO molecule bound to Fe. The whole site is bound to the large subunit via four thiols from strictly conserved Cys residues. In the D. gigas enzyme, the small subunit harbours one [11,14,15]. Also nitrile hydratase exhibits an FTIR band in this region : in its inactive state, this enzyme has an NO bound to Fe giving rise to an FTIR band around 1855 cm Ϫ1 [17].In this study we have examined the FTIR bands exhibited by [Fe]-hydrogenase from D. vulgaris, strain Hildenborough, in more detail. Also the behaviour of the bands under various conditions was studied and compared with that of [NiFe]-hydrogenase from C. vinosum studied under the same conditions. It is concluded that the active site in [Fe]-hydrogenase contains a low-spin Fe with one CN Ϫ and one CO as intrinsic, non-protein ligands. The Fe ion can be either Fe 3ϩ , giving rise to the rhombic 2.10 EPR signal, or Fe 2ϩ (EPR silent). It is proposed that this low-spin Fe is directly linked to a [4Fe-4S] cluster, presumably via two bridging thiols from conserved Cys residues. MATERIALS AND METHODSGrowth of D. vulgaris, subspecies Hildenborough (NCIB 8303), purification of its [Fe]-hydrogenase and determination of purity and activity were performed as described previously [7]. The purity index (A 400 /A 280 ) of the preparation used was 0.36 and the H 2-production activity was 2255 U/mg. Growth of C. vinosum (DSM 185), purification of its [NiFe]-hydrogenase and activity measurements were as previously de-