Tatsuhiko Yagi

Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis

BACKGROUND The hydrogenase of Desulfovibrio sp. catalyzes the reversible oxidoreduction of molecular hydrogen, in conjunction with a specific electron acceptor, cytochrome c3. The Ni-Fe active center of Desulfovibrio hydrogenase has an unusual ligand structure with non-protein ligands. An atomic model at high resolution is required to make concrete assignment of the ligands which coordinate the Ni-Fe center. These in turn will provide insight into the mechanism of electron transfer, during the reaction catalysed by hydrogenase. RESULTS The X-ray structure of the hydrogenase from Desulfovibrio vulgaris Miyazaki has been solved at 1.8 A resolution and refined to a crystallographic R factor of 0.229. The overall folding pattern and the spatial arrangement of the metal centers are very similar to those found in Desulfovibrio gigas hydrogenase. This high resolution crystal structure enabled us to assign the non-protein ligands to the Fe atom in the Ni-Fe site and revealed the presence of a Mg center, located approximately 13 A from the Ni-Fe active center. CONCLUSIONS From the nature of the electron-density map, stereochemical geometry and atomic parameters of the refined structure, the most probable candidates for the four ligands, coordinating the Ni-Fe center, have been proposed to be diatomic S=O, C triple bond O and C triple bond N molecules and one sulfur atom. The assignment was supported by pyrolysis mass spectrometry measurements. These ligands may have a role as an electron sink during the electron transfer reaction between the hydrogenase and its biological counterparts, and they could stabilize the redox state of Fe(II), which may not change during the catalytic cycle and is independent of the redox transition of the Ni. The hydrogen-bonding system between the Ni-Fe and the Mg centers suggests the possible.

Removal of the bridging ligand atom at the Ni–Fe active site of [NiFe] hydrogenase upon reduction with H2, as revealed by X-ray structure analysis at 1.4 Å resolution

BACKGROUND The active site of [NiFe] hydrogenase, a heterodimeric protein, is suggested to be a binuclear Ni-Fe complex having three diatomic ligands to the Fe atom and three bridging ligands between the Fe and Ni atoms in the oxidized form of the enzyme. Two of the bridging ligands are thiolate sidechains of cysteinyl residues of the large subunit, but the third bridging ligand was assigned as a non-protein monatomic sulfur species in Desulfovibrio vulgaris Miyazaki F hydrogenase. RESULTS The X-ray crystal structure of the reduced form of D. vulgaris Miyazaki F [NiFe] hydrogenase has been solved at 1.4 A resolution and refined to a crystallographic R factor of 21.8%. The overall structure is very similar to that of the oxidized form, with the exception that the third monatomic bridge observed at the Ni-Fe site in the oxidized enzyme is absent, leaving this site unoccupied in the reduced form. CONCLUSIONS The unusual ligand structure found in the oxidized form of D. vulgaris Miyazaki F [NiFe] hydrogenase was confirmed in the reduced form of the enzyme, with the exception that the electron density assigned to the monatomic sulfur bridge had almost disappeared. On the basis of this finding, as well as the observation that H2S is liberated from the oxidized enzyme under an atmosphere of H2 in the presence of its electron carrier, it was postulated that the monatomic sulfur bridge must be removed for the enzyme to be activated. A possible mechanism for the catalytic action of the hydrogenase is proposed.

Purification and properties of cytochrome c3 of desulfovibrio vulgaris, miyazaki

Abstract Cytochrome c 3 was isolated in a homogenous state from Desulfovibrio vulgaris , Miyazaki, and its properties examined and compared with those of the cytochrome c 3 from D. vulgaris , Hildenborough. The absorption spectrum of the ultraviolet region of ferrocytochrome c 3 was recorded for the first time. The spectra of the cytochrome c 3 from the Miyazaki strain has five peaks (at 552, 523, 419, 323 and 275 nm) in the ferro-form, three peaks (at 530 with a shoulder at 560, 410 and 350 nm) in the ferri-form, and eight isosbestic points (at 560, 542, 532, 508, 432, 412, 343 and 254 nm). Upon contact with CO, the spectrum of the ferro-form changed. Its isoelectric point is near 10.6, and its redox potential, −0.29 V. The Miyazaki strain cytochrome c 3 contains 4 hemes. Its N-terminus is histidine. Arginine, as well as isoleucine, is absent. These features are all different from those reported for the cytochrome c 3 of D. vulgaris , Hildenborough.

Reversible voltammetric response for a molecule containing four non-equivalent redox sites with application to cytochrome c3 of Desulfovibrio vulgaris, strain Miyazaki

Abstract The cyclic voltammetric and differential pulse polarographic behavior of cytochrome c 3 of Desulfovibrio vulgaris , strain Miyazaki, has been evaluated in terms of a model employing four reversible redox centers. Both types of experiments can be fit by digital simulations using the four standard potentials: E 1 0 =−0.467, E 2 0 =−0.519, E 3 0 =−0.539 and E 4 0 =−0.580 V vs. SCE. The results are interpreted to mean that the four redox centers are chemically different and only weakly interacting. The relationships between the observed macroscopic standard potentials and the microscopic standard potentials for reduction of individual sites are discussed.

Significance of hydrogen burst from growing cultures of Desulfovibrio vulgaris, Miyazaki, and the role of hydrogenase and cytochrome c3 in energy production system

In an early stage of the growth of Desulfovibrio vulgaris, Miyazaki, a burst of H2 occurred, and lasted for a few hours. The H2S production which paralleled the cell proliferation was very low in the H2 burst period, and began to increase thereafter. Hydrogenase (hydrogen: ferricytochrome c3 oxidoreductase, EC1. 12.2.1), cytochrome c3 and desulfoviridin also increased after the H2 burst. These phenomena were common to all the cultural conditions tested, i.e., the cell growth is always preceded by the initial H2 burst. Hydrogenase of cells harvested in the H2 burst peroid was composed mainly of the high molecular weight species (mol. wt., 180,000), whereas that of the cells harvested later was composed of both the high molecular weight and the low molecular weight (mol. wt., 70,000) species. It was suggested that the former enzyme was acting as a catalyzer in the initial H2 burst to effect the substrate level phosphorylation during the breakdown of lactate to acetate and CO2, whereas the latter was induced by the H2 produced by the cells themselves to recycle H2 in order to supply electrons to the reducing system of sulfur oxy-acids coupled to electron transfer phosphorylation. The amount of cytochrome c3 in cells harvested from an iron-deficient medium was as high as that in cells harvested from an iron-rich medium, suggesting the significance of this electron carrier in the cellular metabolism.

EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: Comparison with the NiFe enzyme from Desulfovibrio gigas

We have carried out a detailed redox titration monitored by EPR on the hydrogenase from Desulfovibrio vulgaris Miyazaki. Typical 3Fe and nickel signals have been observed, which are very similar to those given by Desulfovibrio gigas hydrogenase in all the characteristic redox states of the enzyme. This confirms that D. vulgaris Miyazaki hydrogenase is a Ni-Fe enzyme closely related to that from D. gigas, as was recently proposed on the basis of sequence comparisons (Deckers, H.M., Wilson, F.R. and Voordouw, G. (1990) J. Gen. Microb. 136, 2021-2028).

Purification and properties of cytochrome c-553, an electron acceptor for formate dehydrogenase of Desulfovibrio vulgaris, Miyazaki

Cytochrome c-553 of Desulfovibrio vulgaris, Miyazaki, was purified to homogeneity. The absorption spectrum of the ferro form has four peaks at 553, 525, 417 and 317 nm with a plateau near 280 nm, and that of the ferri form has three peaks at 525, 410 and 360 nm with a plateau near 280 nm and a shoulder at 560 nm. The millimolar absorbance coefficient of the alpha-peak of the ferro form is 23.9. The molecular weight of cytochrome c-553 is 8000, and it contains one heme. Its isoelectric point is rather alkaline, and its standard redox potential is -0.26 V at pH 7.0. Its amino acid composition is unique; it lacks proline, isoleucine and tryptophan. Ferrocytochrome c-553 does not combine with CO, nor does it transfer electrons directly to various redox carriers such as flavin nucleotides, methylene blue, indigodisulfonate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, viologens and cytochrome c3, but is oxidized by ferricyanide or by O2. Cytochrome c-553 can be reduced by formate dehydrogenase of this bacterium in the presence of formate, but not by hydrogenase under H2. The formate dehydrogenase does not reduce cytochrome c3 in the presence of formate. The systematic name for formate dehydrogenase of D. vulgaris is, therefore, established as formate:ferricytochrome c-553 oxidoreductase in EC subclass 1.22.-.

Electrical conduction of hemoprotein in the solid phase: Anhydrous cytochrome c3 film

Cytochrome c3 can be reduced with molecular hydrogen under the action of hydrogenase [hydrogen: ferricytochrome c3 oxidoreductase, EC 1.12.2.1] even in the solid state. The electrical conductivity of a cytochrome c3 anhydrous film containing a trace amount of hydrogenase was measured at physiological temperatures as a function of temperature and hydrogen pressure. Ferricytochrome c3 (oxidized form) and ferrocytochrome c3 (reduced form) equilibrate at a given temperature and pressure by a catalytic action of hydrogenase. Under these conditions, the conductivity of cytochrome c3 showed an unusual temperature dependence: The activation energy was positive under higher hydrogen pressure, but was negative under lower pressure. These findings are interpreted as a thermal equilibration between the ferri‐ and ferro‐ forms using the Hill equation for the reduction ratio and applying the theory of semiconduction to the electrical conductivity. The theory predicted that the activation energy of conductivity would co...

Isolation and crystallization of high molecular weight cytochrome from Desulfovibrio valgaris Hildenborough

Abstract High molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough has been isolated, purified and crystallized. The molecular weight was estimated to be 75 000 as the mean value from the results of gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains 16 c-type heme groups per molecule. The purified high molecular weight cytochrome c shows typical c-type cytochrome absorption spectra, with an α-peak at 553.2 nm (ϵ553.2mM = 428) in the ferro form. The amino acid composition shows that the protein contains a sufficient number of cysteine and histidine residues to account for the high content of heme groups. The amino acid composition of D. vulgaris Hildenborough high molecular weight cytochrome c has no similarities to the D. desulfuricans hexahemoprotein nitrite reductase. The high content of the heme groups might suggest that the cytochrome has an alternate intrinsic biological function. Crystals of the high molecular weight cytochrome c have been grown from solutions of poly(ethylene glycol) 1000 or 2-methyl-2,4-pentanediol. The crystals are in space group P62 or P64 with unit cell dimensions a = b = 227.8, c = 105.7 A and γ = 120°. A large unit cell volume of 4.75 · 106 A3 suggests that there are four or five protein molecules per asymmetric unit. They diffract to better than 4.0 A resolution and appear to be resistant to radiation damage.

EPR redox study of cytochrome c3 from Desulfovibrio vulgaris Miyazaki

We report the results of an EPR potentiometric titration of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki: the EPR spectral features of the four hemes are identified. The four midpoint redox potentials, which are deduced from the integrated intensity variations as a function of the redox potential, are within the range −230 to −360 mV with two nearly equal intermediate values, in agreement with previous electrochemical measurements. A structural change of the environment of the heme with the most negative potential is observed during the first step of the reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration, and the hemes in the tridimensional structure, is discussed.