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 Km 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.

Intermediates in the Catalytic Cycle of [NiFe] Hydrogenase: Functional Spectroscopy of the Active Site

“…[15] The [NiFe] hydrogenase involved in this anaerobic respiration is a periplasmic enzyme attached to the membrane with a tetrahaem cyt c module as its physiological electron acceptor. [16,17] It has a molecular weight of approximately 90 kDa and consists of two subunits; [18] the large subunit contains the hetero-bimetallic [NiFe] centre (known as the active site) and the small subunit contains three almost linearly arranged iron-sulphur cofactors, one [Fe 3 S 4 ] cluster in-between two low-potential [Fe 4 S 4 ] clusters, that are involved in the electron transfer from and to the active site [19] ( Figure 1). Proton transfer involves several possible pathways.…”

“…[54] X-ray crystallographic experiments on the oxidised enzyme detected additional electron density in the bridging position between the two metals. EPR experiments with 17 O 2 -labelled gas [58] and H 2 17 O-enriched water [59] have shown that an oxygen species is present in the active site of both oxidised states that can be derived either from the gas or the solvent water.…”

Effects of Mesoporous Structures on Direct Electron Transfer-Type Bioelectrocatalysis: Facts and Simulation on a Three-Dimensional Model of Random Orientation of Enzymes

“…25 When we assume a uniform orientation of enzymes, we analyze steady-state voltammograms using Eqs. (1) or (21). Analyzing the steady-state voltammograms of BOD, the values of k c /k (= k c /k°exp(¹¢d )) are estimated about 1-100.…”

“…21 All other chemicals were of analytical regent grade, and solutions were prepared with distilled water.…”

Incorporation of Unnatural Amino Acids into Cytochrome c3 and Specific Viologen Binding to the Unnatural Amino Acid

“…The 3D structure of cyA C H T U N G T R E N N U N G toA C H T U N G T R E N N U N G chrome c 3 is shown in Figure 1, however, mechanisms for inter-or intramolecular electron transfer through the four hemes have not yet been clarified. [13][14][15][16][17] A site-specific modification of cyA C H T U N G T R E N N U N G to-A C H T U N G T R E N N U N G chrome c 3 with a redox mediator is a suitable method for investigating the role of each heme, because the redox mediator donates an electron to and accepts an electron from a heme near to where it binds.…”