Single crystals of hydrogenase from Desulfovibrio vulgaris Miyazaki F

The hydrogenase solubilized from the particulate fraction from Desulfovibrio vulgaris Miyazaki F (IAM 12604) has been crystallized. Although the solubilized hydrogenase purified by the previous method (Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S., and Inokuchi, H. (1976) J. Biochem. (Tokyo) 79,661-671) revealed a single band upon disc electrophoresis, it could not be crystallized. The apparently homogeneous hydrogenase has been separated into three components of similar molecular weights by high performance liquid chromatography on DEAE-Toyopearl. Each hydrogenase component was successfully crystallized by means of the vapor diffusion method with polyethylene glycol or 2-methyl-2,4-pentanediol as a precipitating agent. Seeding procedure is necessary to grow an x-ray grade crystal. Preliminary x-ray experiments reveal that crystals grown from one component are in space group of P2(1)2(1)2(1) with a = 102.1(1), b = 126.8 (3), and c = 66.9(1) A. The unit cell volume of 8.66 X 10(5) A3 suggests that it contains one molecule/asymmetric unit (Vm = 2.43). The crystals grown from another component are in the same space group with a = 99.6(1), b = 126.8(3), c = 66.9(1) A, and the unit cell volume is 8.45 X 10(5) A3 (Vm = 2.37). The crystals diffract more than 2.5 A and are suitable for complete crystal analysis. Up to 4 A resolution native data have been collected on a diffractometer.     

Extraction of periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Abstract Periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) was extracted according to the method of van der Westen [8] and the effect of trace minerals on the extractability of this enzyme was investigated. The final growth yields in the presence or absence of trace minerals were the same; however, the growth was much faster and the amount of periplasmic hydrogenase extracted was significantly lower in the presence of trace minerals. Polyacrylamide gel electrophoresis showed the presence of 2 hydrogenases in D. vulgaris , one soluble and the other possibly membrane-bound.     

EPR-detectable redox centers of the periplasmic hydrogenase from Desulfovibrio vulgaris

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.     

Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.     

Characterization of the periplasmic hydrogenase from Desulfovibrio gigas.

The hydrogenase of the sulfate-reducer $\text{Desulfovibrio gigas}$ has been purified to homogeneity. The pure enzyme shows a specific activity of 90 μmoles H₂ evolved/min./mg protein. Its molecular weight is 89,500 and its is composed of two different subunits (mol. wt. : 62,000 and 26,000) which are not covalently bound. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein. The millimolar extinction coefficients of the hydrogenase are 46.5 and 170 respectively at 400 and 280 nm. It contains about 12 iron atoms and 12 acid-labile sulfur groups per molecule and the quantitative extrusion of the Fe-S centers of the hydrogenase indicates the presence of 3 Fe₄S₄ clusters. This hydrogenase has 21 half-cystine residues per molecule and a preponderance of aromatic amino-acids.     

Properties of purified hydrogenase from the particulate fraction of Desulfovibrio vulgaris, Miyazaki.

The properties of purified hydrogenase [EC 1.12.2.1] solubilized from particulate fraction of sonicated Desulfovibrio vulgaris cells are described. The enzyme was a brownish iron-sulfur protein of molecular weight 89,000, composed of two different subunits (mol. wt.: 28,000 and 59,000), and it contained 7-9 iron atoms and 7-8 labile sulfide ions. Molybdenum was not detected in the preparation. The absorption spectrum of the enzyme was characteristic of iron-sulfur proteins. The millimolar absorbance coefficients of the enzyme were about 164 at 280nm, and 47 at 400nm. The absorption spectrum of the enzyme in the visible region changed upon incubating the enzyme under H2 in the presence of cytochrome c3, but not in its absence. This spectral change was due to the reduction of the enzyme. The absorbance ratio at 400nm of the reduced and the oxidized forms of the enzyme was 0.66. The activity of the enzyme was hardly affected by metal-complexing agents such as cyanide, azide, 1,10-phenanthroline, etc., except for CO, which was a strong inhibitor of the enzyme. The activity was inhibited by SH-reagents such as p-chloromercuribenzenesulfonate. The enzyme was significantly resistant to urea, but susceptible to sodium dodecyl sulfate. These properties were very similar to those of clostridial hydrogenase [EC 1.12.7.1], in spite of differences in the acceptor specificity and subunit structure.     

Analysis of the active center of hydrogenase from Desulfovibrio vulgaris Miyazaki by magnetic measurements

Hydrogenase [hydrogen: ferricytochrome c3 oxidoreductase, EC 1.12.2.1] solubilized and purified from the particulate fraction of Desulfovibrio vulgaris Miyazaki F (IAM 12604) contains 8 iron and 8 labile sulfide ions in one molecule which is composed of two unequal subunits (Mr: 60,000 + 29,000). It does not contain nickel atoms. The EPR (electron paramagnetic resonance) spectrum has an isotropic signal at g = 2.017 which is independent of the temperature. The peak-to-peak width of the signal is about 20 G. The signal intensity is nearly equivalent to 1 unpaired electron per molecule. No other signals can be detected in the field range between 2,240 and 4,240 G (which corresponds to g-values between 2.91 and 1.54). Ferricyanide has only a little effect on the shape and intensity of the EPR signal. The hydrogenase reduced under H2 is EPR silent. The M?ssbauer spectrum has no hyperfine splitting at 4K. The isomer shift and quadrupole splitting at 77K are 0.38 and 0.87 mm/s, respectively. Based on these magnetic measurements, the structure of the active center of hydrogenase was suggested to be [4Fe-4S]3+ + [4Fe-4S]2+.     

Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F

A hydrogenase operon was cloned from chromosomal DNA isolated from Desulfovibrio vulgaris Miyazaki F with the use of probes derived from the genes encoding [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough. The nucleic acid sequence of the cloned DNA indicates this hydrogenase to be a two-subunit enzyme: the gene for the small subunit (267 residues; molecular mass = 28763 Da) precedes that for the large subunit (566 residues; molecular mass = 62495 Da), as in other [NiFe] and [NiFeSe] hydrogenase operons. The amino acid sequences of the small and large subunits of the Miyazaki hydrogenase share 80% homology with those of the [NiFe] hydrogenase from Desulfovibrio gigas. Fourteen cysteine residues, ten in the small and four in the large subunit, which are thought to co-ordinate the iron-sulphur clusters and the active-site nickel in [NiFe] hydrogenases, are found to be conserved in the Miyazaki hydrogenase. The subunit molecular masses and amino acid composition derived from the gene sequence are very similar to the data reported for the periplasmic, membrane-bound hydrogenase isolated by Yagi and coworkers, suggesting that this hydrogenase belongs to the general class of [NiFe] hydrogenases, despite its low nickel content and apparently anomalous spectral properties.     

EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni-Fe 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).     

Spectroelectrochemical characterization of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F

The active site in the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F has been investigated by Fourier transform infrared (FTIR) spectroscopy. Analysis of the spectra allowed the three diatomic inorganic ligands to Fe in this enzyme to be identified as one CO molecule and two CN(-) molecules. Furthermore, pH-dependent redox titrations were performed to determine the midpoint potentials as well as the pK value of the respective reactions and revealed that each single-electron redox transition is accompanied by a single-proton transfer step. The comparison of these spectra with those published for other [NiFe] hydrogenases shows that the electronic structure of the active sites of these enzymes and their redox processes are essentially the same. Nevertheless, differences with respect to the frequency of the CO band and the pH dependence of the Ni-R states have been observed. Finally, the frequency shifts of the bands in the IR spectra were interpreted with respect to the electronic configuration of the redox intermediates in the catalytic cycle.     

Redox interaction of cytochrome c3 with [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

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.     

Putative signal peptide on the small subunit of the periplasmic hydrogenase from Desulfovibrio vulgaris.

We sequenced the NH2 terminus of the large and small subunits of the periplasmic hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) and found that the small subunit lacks a region of 34 NH4-terminal amino acids coded by the gene for the small subunit (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). We suggest that this region constitutes a signal peptide based on comparison with known procaryotic signal peptides.     

Magnetic susceptibility of hydrogenase from Desulfovibrio vulgaris

Magnetization and magnetic susceptibility measurements revealed that the hydrogenase [EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki F has an independent unpaired electron in its iron-sulfur cluster. The paramagnetic center of the Desulfovibrio hydrogenase is, therefore, different from that in the Chromatium hydrogenase which interacts with another paramagnetic center, probably nickel.     

Characterization and complete amino acid sequence of ferredoxin II from Desulfovibrio vulgaris Miyazaki

One of 2 ferredoxins, Fd II, purified from Desulfovibrio vulgaris Miyazaki (DvM) has been characterized and its complete amino acid sequence established. Fd II is composed of 63 amino acid residues and contains 7 cysteinyl residues but has only 4 iron atoms in an iron-sulfur cluster of a standard redox potential of -405 mV. The arrangement of cysteinyl residues in the protein suggests that some cysteinyl residues are not directly involved in ligation to the iron-sulfur cluster. Homology is recognized among Fd II (DvM), Fd I (D. desulfuricans Norway), Fd I (D. africanus Benghazi), and Fd (D. gigas). Although Fd I and Fd II in DvM are poorly homologous, the C-terminal half of Fd I is fairly homologous to the N-terminal half of Fd II. Fd II is 40% as effective as Fd I as an electron carrier for pyruvate dehydrogenase coupled with hydrogenase and cytochrome c3.     

Purification and characterization of ferredoxin from Desulfovibrio vulgaris Miyazaki

Two ferredoxins, Fd I and Fd II, were isolated and purified from Desulfovibrio vulgaris Miyazaki. The major component, Fd I, is an iron-sulfur protein of Mr 12,000, composed of two identical subunits. The absorption spectra of Fd I and Fd II have a broad absorption shoulder near 400 nm characteristic of iron-sulfur proteins. The purity index, A400/A280, of Fd I is 0.69, and its millimolar absorption coefficient at 400 nm is 3.73 per Fe. It contains two redox centers with discrete redox behaviors. The amino acid composition and the N-terminal sequence of Fd I are similar to those of Fd III of Desulfovibrio africanus Benghazi and Fd II of Desulfovibrio desulfuricans Norway. Fd I does not serve as an electron carrier for the hydrogenase of D. vulgaris Miyazaki, but it serves as a carrier for pyruvate dehydrogenase of this bacterium. The evolution of H2 from pyruvate was observed by a reconstructed system containing purified hydrogenase, cytochrome C3, Fd I, partially purified pyruvate dehydrogenase, and CoA. The H2-sulfite reducing system can be reconstructed from the purified hydrogenase, cytochrome C3, Fd I and desulfoviridin (sulfite reductase), but the reaction rate is very slow compared to that of the crude extract at the same molar ratio of the components.     

Photosensitivity of the Ni-A state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F with visible light

[NiFe] hydrogenase catalyzes reversible oxidation of molecular hydrogen. Its active site is constructed of a hetero dinuclear Ni–Fe complex, and the oxidation state of the Ni ion changes according to the redox state of the enzyme. We found that the Ni-A state (an inactive unready, oxidized state) of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) is light sensitive and forms a new state (Ni-AL) with irradiation of visible light. The Fourier transform infrared (FT-IR) bands at 1956, 2084 and 2094 cm^?1 of the Ni-A state shifted to 1971, 2086 and 2098 cm^?1 in the Ni-AL state. The g-values of g_x = 2.30, g_y = 2.23 and g_z = 2.01 for the signals in the electron paramagnetic resonance (EPR) spectrum of the Ni-A state at room temperature varied for ?0.009, +0.012 and +0.010, respectively, upon light irradiation. The light-induced Ni-AL state converted back immediately to the Ni-A state under dark condition at room temperature. These results show that the coordination structure of the Fe site of the Ni-A state of [NiFe] hydrogenase is perturbed significantly by light irradiation with relatively small coordination change at the Ni site.     

Selenium is involved in regulation of periplasmic hydrogenase gene expression in Desulfovibrio vulgaris Hildenborough

Desulfovibrio vulgaris Hildenborough is a good model organism to study hydrogen metabolism in sulfate-reducing bacteria. Hydrogen is a key compound for these organisms, since it is one of their major energy sources in natural habitats and also an intermediate in the energy metabolism. The D. vulgaris Hildenborough genome codes for six different hydrogenases, but only three of them, the periplasmic-facing [FeFe], [FeNi]1, and [FeNiSe] hydrogenases, are usually detected. In this work, we studied the synthesis of each of these enzymes in response to different electron donors and acceptors for growth as well as in response to the availability of Ni and Se. The formation of the three hydrogenases was not very strongly affected by the electron donors or acceptors used, but the highest levels were observed after growth with hydrogen as electron donor and lowest with thiosulfate as electron acceptor. The major effect observed was with inclusion of Se in the growth medium, which led to a strong repression of the [FeFe] and [NiFe]1 hydrogenases and a strong increase in the [NiFeSe] hydrogenase that is not detected in the absence of Se. Ni also led to increased formation of the [NiFe]1 hydrogenase, except for growth with H2, where its synthesis is very high even without Ni added to the medium. Growth with H2 results in a strong increase in the soluble forms of the [NiFe]1 and [NiFeSe] hydrogenases. This study is an important contribution to understanding why D. vulgaris Hildenborough has three periplasmic hydrogenases. It supports their similar physiological role in H2 oxidation and reveals that element availability has a strong influence in their relative expression.     

Amino acid sequence of ferredoxin I from Desulfovibrio vulgaris Miyazaki

The amino acid sequence of ferredoxin (Fd) I, purified from Desulfovibrio vulgaris Miyazaki, has been established. Fd I is strikingly similar to Fd III of D. africanus Benghazi with 84% homology. Both have the sequence, -Cys-x-x-Asp-x-x-Cys-x-x-x-Cys-Pro- in the N-terminal half, and the sequence, -Cys-x-x-Cys-x-x-Cys-x-x-x-Cys-Glu- in the C-terminal half of the molecule, instead of the common sequences for ligation to the usual [4Fe-4S] clusters. Fd I has 76% homology to Fd II of D. desulfuricans Norway.     

Crystallographic study of cytochrome c553 from Desulfovibrio vulgaris Miyazaki

Cytochrome c553 from the sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki, has been crystallized. The combination of microdialysis and vapor diffusion allowed successful crystallization. The crystals were of good quality, and useful data were obtained that extended to the nominal resolution of 1.3 A. The space group is P4(3)2(1)2 with cell dimensions of a = b = 42.7 A, c = 103.4 A. More than twenty heavy-atom reagents were screened with the isomorphous replacement technique, and only the mersalyl derivative could be used for the phase determination. The single isomorphous replacement method combined with the anomalous scattering effect of the Hg-atom in mersalyl and the Fe-atom of the heme group was used for the phase determination.     

Single crystal EPR studies of the oxidized active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

The Ni-A and the Ni-B forms of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F have been studied in single crystals by continuous wave and pulsed EPR spectroscopy at different temperatures (280?K, 80?K, and 10?K). For the first time, the orientation of the g-tensor axes with respect to the recently published atomic structure of the active site at 1.8?Å resolution was elucidated for Ni-A and Ni-B. The determined g-tensors have a similar orientation. The configuration of the electronic ground state is proposed to be Ni(III) 3d ¹ _z2 for Ni-A and Ni-B. The g _z principal axis is close to the Ni-S(Cys549) direction; the g _x and the g _y axes are approximately along the Ni-S(Cys546) and Ni-S(Cys81) bonds, respectively. It is proposed that the difference between the Ni-A and Ni-B states lies in a protonation of the bridging ligand between the Ni and the Fe.