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Hydrogenases catalyze the reversible oxidation of molecular hydrogen. The active site of the FeFe hydrogenases (H-cluster) contains a catalytically active binuclear subcluster (2Fe(H)) connected to a "cubane" 4Fe4S(H) subcluster. Here we present an IR spectroelectrochemical study of the FeFe hydrogenase HydA1 isolated from the green alga Chlamydomonas reinhardtii. The enzyme shows IR bands similar to those observed for bacterial FeFe hydrogenases. They are assigned to the stretching vibrations of the CN(-) and CO ligands on both irons of the 2Fe(H) subcluster. By following changes in frequencies of the IR bands during electrochemical titrations, two one-electron redox processes of the active enzyme could be distinguished. The reduction of the oxidized state (H(ox)) occurred at a midpoint potential of -400 mV vs NHE (H(ox)/H(red) transition) and relates to a change of the formal oxidation state of the binuclear subcluster. A subsequent reduction (H(red)/H(sred) transition) was determined to have a midpoint potential of -460 mV vs NHE. On the basis of the IR spectra, it is suggested that the oxidation state of the binuclear subcluster does not change in this transition. Tentatively, a reduction of the 4Fe4S(H) cluster has been proposed. In contrast to the bacterial FeFe hydrogenases, where the bridging CO ligand becomes terminal when going from H(ox) to H(red), in HydA1 the bridging CO is present in both the H(ox) and H(red) state. The removal of the bridging CO moiety has been observed in the H(red) to H(sred) transition. The significance of this result for the hydrogen conversion mechanism of this class of enzymes is discussed.
Silakov et al. (Mon,) studied this question.