Redox Interaction of Cytochromec3with [NiFe] Hydrogenase fromDesulfovibrio vulgarisMiyazaki F

Abstract

Cytochrome c₃ isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between cytochrome c₃ and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly ¹⁵N-labeled ferric cytochrome c₃ except N-terminus, Pro, and Gly73 were assigned. ¹H-¹⁵N HSQC spectra were recorded for ¹⁵N-labeled ferric and ferrous cytochrome c₃, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes 1 and 3 in ferrous cytochrome c₃ was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous cytochrome c₃. To investigate the transient complex of cytochrome c₃ and hydrogenase in detail, modeling of the complex was performed for the oxidized proteins using a docking program, ZDOCK 2.3, and NMR data. Furthermore, the roles of lysine residues of cytochrome c₃ in the interaction with hydrogenase were investigated by site-directed mutagenesis. When the lysine residues around heme 4 were replaced by an uncharged residue, methionine, one by one, the K_m of the electron-transfer kinetics increased. The results showed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 are important for formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome c₃ reduction. This finding is consistent with the most possible structure of the transient complex obtained by modeling.