Spectroelectrochemical characterization of the active site of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii

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 theCN^- andCO 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/Hs_red 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 bridgingCOligand 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.