Silakov Alexey, Kamp Christina, Reijerse Eduard, Happe Thomas, Lubitz Wolfgang
Max-Planck-Institut für Bioanorganische Chemie, Stiftstr. 34-36, 45470 Mülheim an der Ruhr, Germany.
Biochemistry. 2009 Aug 25;48(33):7780-6. doi: 10.1021/bi9009105.
Hydrogenases catalyze the reversible oxidation of molecular hydrogen. The active site of the [FeFe] hydrogenases (H-cluster) contains a catalytically active binuclear subcluster (2Fe) connected to a "cubane" 4Fe4S 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 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 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.
氢化酶催化分子氢的可逆氧化反应。[FeFe]氢化酶（H-簇）的活性位点包含一个与“立方烷”4Fe4S亚簇相连的具有催化活性的双核亚簇（2Fe）。在此，我们展示了对从绿藻莱茵衣藻中分离出的[FeFe]氢化酶HydA1的红外光谱电化学研究。该酶显示出与细菌[FeFe]氢化酶中观察到的相似的红外吸收带。它们被归属于2Fe亚簇中两个铁原子上的CN(-)和CO配体的伸缩振动。通过跟踪电化学滴定过程中红外吸收带频率的变化，可以区分活性酶的两个单电子氧化还原过程。氧化态（H(ox)）的还原发生在相对于标准氢电极（NHE）为-400 mV的中点电位（H(ox)/H(red)转变），并且与双核亚簇的形式氧化态变化有关。随后的还原（H(red)/H(sred)转变）被确定相对于NHE的中点电位为-460 mV。基于红外光谱，表明在该转变过程中双核亚簇的氧化态没有变化。初步推测，已提出4Fe4S簇发生了还原。与细菌[FeFe]氢化酶不同，在从H(ox)转变为H(red)时桥连的CO配体变为端基配体，而在HydA1中，桥连的CO在H(ox)和H(red)状态下均存在。在H(red)到H(sred)转变中观察到了桥连CO部分的去除。讨论了该结果对于这类酶的氢转化机制的意义。
