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The ferriheme proteins nitrophorin 4 and 7 convert nitrite into nitric oxide at neutral pH, which is an unprecedented case among the proteins. The initial complex formed is a 6-coordinate low-spin Fe(III)-eta(1)-N nitro complex (S = 1/2). Determination of the reaction kinetics of the initial nitrite binding step allows the estimation of the nitrite affinity being comparable to that of met-hemoglobin and met-myoglobin, which indicates that nitrite may be a substrate of nitrophorins in vivo.

The [NiFe] hydrogenase from the sulphate-reducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SIr, provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SIa, Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SIr was found to be light-sensitive (T a parts per thousand currency sign 110 K), similar to the active Ni-C and the CO-inhibited states. Transition to the final photoproduct state (Ni-SL) was shown to involve an additional transient light-induced state (Ni-SI1961). Rapid scan kinetic infrared measurements provided activation energies for the transition from Ni-SL to Ni-SIr in protonated as well as in deuterated samples. The inhibitor CO was found not to react with the active site of the Ni-SL state. The wavelength dependence of the Ni-SIr photoconversion was examined in the range between 410 and 680 nm. Light-induced effects were associated with a nickel-centred electronic transition, possibly involving a change in the spin state of nickel (Ni2+). In addition, at T a parts per thousand currency sign 40 K the CN- stretching vibrations of Ni-SL were found to be dependent on the colour of the monochromatic light used to irradiate the species, suggesting a change in the interaction of the hydrogen-bonding network of the surrounding amino acids. A possible mechanism for the photochemical process, involving displacement of the oxygen-based ligand, is discussed.

Ribonucleotide reduction, the unique step in DNA-precursor biosynthesis, involves radical-dependent redox chemistry and diverse metallo-cofactors. The metallo-cofactor (R2F) encoded by the nrdF (nucleotide reduction) gene in Corynebacterium ammoniagenes ATCC 6872 was isolated after homologous expression and a new crystal form of ribonucleotide reductase R2F was obtained. R2F was crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. A data set was collected to 1.36 angstrom resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group C2, with unit-cell parameters a = 96.21, b = 87.68, c = 83.25 angstrom, beta = 99.29 degrees. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient (V(M)) of 2.69 angstrom(3) Da(-1); the solvent content was estimated to be 54.3%. X-ray fluorescence spectroscopy and MAD diffraction data indicated the presence of manganese in the molecule and the absence of iron.

Light-oxygen-voltage (LOV) proteins play an important role in blue-light-dependent physiological processes in many organisms. The LOV domain of the blue-light receptor YtvA from Bacillus amyloliquefaciens FZB42 has been purified and crystallized at 277 Kusing the sitting-drop vapour-diffusion method with 2-ethoxyethanol as a precipitant. A data set was collected to 1.60 angstrom resolution from a single crystal at 100 K using synchrotron radiation. The LOV domain of YtvA crystallized in space group C222(1), with unit-cell parameters a = 64.95, b = 83.76, c = 55.81 angstrom. The crystal structure of the LOV domain of YtvA was determined by the molecular-replacement method. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (V-M) of 3.04 angstrom(3)Da(-1); the solvent content was estimated to be 59.5%.

[NiFe] hydrogenases catalyze the reversible oxidation of dihydrogen. For this simple reaction the molecule has developed a complex catalytic mechanism, during which the enzyme passes through various redox states. The [NiFe] hydrogenase contains several metal centres, including the bimetallic Ni-Fe active site, iron-sulfur clusters and a Mg2+ ion. The Ni-Fe active site is located in the inner part of the protein molecule, therefore a number of pathways are involved in the catalytic reaction route. These consist of an electron transfer pathway, a proton transfer pathway and a gas-access channel. Over the last 10-15 years we have been investigating the crystal structures of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F, which is a sulfate-reducing anaerobic bacterium. So far the crystal structures of the oxidized, H-2-reduced and carbon monoxide inhibited states have been determined at high resolution and have revealed a rather unique structure of the hetero-bimetallic Ni-Fe active site. Furthermore, intensive spectroscopic studies have been performed on the enzyme. Based on the crystal structure, a water-soluble Ni-Ru complex has been synthesized as a functional model for the [NiFe] hydrogenases. The present review gives an overview of the catalytic reaction mechanism of the [NiFe] hydrogenases.

Sulfur in its various oxidation states is used for energy conservation in many microorganisms. Adenylylsulfate reductase is a key enzyme in the sulfur-reduction pathway of sulfate-reducing bacteria. The adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the vapour-diffusion method with ammonium sulfate as the precipitating agent. A data set was collected to 1.7 angstrom resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 125.93, c = 164.24 angstrom. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient (V-M) of 4.02 angstrom(3) Da(-1); the solvent content was estimated to be 69.4%.

The membrane-bound [NiFe] hydrogenase is a unique metalloprotein that is able to catalyze the reversible oxidation of hydrogen to protons and electrons during a complex reaction cycle. The [NiFe] hydrogenase was isolated from the photosynthetic purple sulfur bacterium Allochromatium vinosum and its crystallization and preliminary X-ray analysis are reported. It was crystallized by the hanging-drop vapour-diffusion method using sodium citrate and imidazole as crystallization agents. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 205.00, b = 217.42, c = 120.44 angstrom. X-ray diffraction data have been collected to 2.5 angstrom resolution.

Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of β-catenin by glycogen synthase kinase 3β. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P61 or P65, with unit-cell parameters a = b = 91.49, c = 84.92 Å. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 Å. © International Union of Crystallography 2007.

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 N-15-labeled ferric cytochrome C3 except N-terminus, Pro, and Gly73 were assigned. H-1-N-15 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 I 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.

Complicated pH-properties of the tetraheme cytochrome c₃ (cyt c₃) from Desulfovibrio vulgaris Miyazaki F (DvMF) were examined by the pH titrations of ¹H-¹⁵N HSQC spectra in the ferric and ferrous states. The redox-linked pK_a shift for the propionate group at C13 of heme 1 was observed as the changes of the NH signals around it. This pK_a shift is consistent with the redox-linked conformational alteration responsible for the cooperative reduction between hemes 1 and 2. On the other hand, large chemical shift changes caused by the protonation/deprotonation of Glu41 and/or Asp42, and His67 were redox-independent. Nevertheless, these charged residues affect the redox properties of the four hemes. Furthermore, one of interesting charged residues, Glu41, was studied by site-directed mutagenesis. E41K mutation increased the microscopic redox potentials of heme 1 by 46 and 34 mV, and heme 2 by 35 and 30 mV at the first and last reduction steps, respectively. Although global folding in the crystal structure of E41K cyt c₃ is similar to that of wild type, local change was observed in ¹H NMR spectrum. Glu41 is important to keep the stable conformation in the region between hemes 1 and 2, controlling the redox properties of DvMF cyt c₃. In contrast, the kinetic parameters for electron transfer from DvMF [NiFe] hydrogenase were not influenced by E41K mutation. This suggests that the region between hemes 1 and 2 is not involved in the interaction with [NiFe] hydrogenase, and it supports the idea that heme 4 is the exclusive entrance gate to accept the electron in the initial reduction stage.<br>

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the beta-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH- bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the "ready" state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the "unready" state of the enzyme.

Hydrogenases catalyze oxidoreduction of molecular hydrogen and have potential applications for utilizing dihydrogen as an energy source. [NiFe] hydrogenase has two different oxidized states, Ni-A (unready, exhibits a lag phase in reductive activation) and Ni-B (ready). We have succeeded in converting Ni-B to Ni-A with the use of Na2S and O-2 and determining the high-resolution crystal structures of both states. Ni-B possesses a monatomic nonprotein bridging ligand at the Ni-Fe active site, whereas Ni-A has a diatomic: species. The terminal atom of the bridging species of Ni-A occupies a similar position as C of the exogenous CO in the CO complex (inhibited state). The common features of the enzyme structures at the unready (Ni-A) and inhibited (CO complex) states are proposed. These findings provide useful information on the design of new systems of biomimetic dihydrogen production and fuel cell devices.

The catalytic domain of death-associated protein kinase (DAPK) has been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with PEG 8000 and magnesium acetate as precipitants. Complexes with the inhibitor staurosporine and its analogue BDB402 were also crystallized in the presence of PEG 400 and PEG 8000, respectively. Diffraction data were collected to 2.4 Angstrom for the native catalytic domain, to 2.9 Angstrom for the staurosporine complex and to 2.7 Angstrom for the BDB402 complex. All three crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a=77.992, b=109.909, c=50.063 Angstrom for the catalytic domain, a=78.911, b=113.162, c=50.658 Angstrom for the staurosporine complex and a=77.337, b=108.869, c=50.186 Angstrom for the BDB402 complex. In both complexes the inhibitor molecule was clearly assigned in the difference Fourier map calculated on the basis of the phases obtained from the structure of the catalytic domain.

The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 2.1-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene (eno) and part of the methylenetetrahydrofolate dehydrogenase gene (folD). The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids. An expression system for eno under control of the T7 promoter was constructed in E. coli. The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of D-(+)-2-phosphoglyceric acid (2-PGA). The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied. The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D. vulgaris was grown on lactate. (C) 2003 Elsevier B.V. All rights reserved.

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.

In the catalytic cycle of [NiFe] hydrogenase the paramagnetic Ni-C intermediate is of key importance, since it is believed to carry the substrate hydrogen, albeit in a yet unknown geometry. Upon illumination at low temperatures, Ni-C is converted to the so-called Ni-L state with markedly different spectroscopic parameters. It is suspected that Ni-L has lost the "substrate hydrogen". In this work, both paramagnetic states have been generated in single crystals obtained from the [NiFe] hydrogenase from Desulfovibrio, vulgaris Miyazaki F. Evaluation of the orientation dependent spectra yielded the magnitudes of the g tensors and their orientations in the crystal axes system for both Ni-C and Ni-L. The g tensors could further be related to the atomic structure by comparison with the X-ray crystallographic structure of the reduced enzyme. Although the g tensor magnitudes of Ni-C and Ni-L are quite different, the orientations of the resulting g tensors are very similar but differ from those obtained earlier for Ni-A and Ni-B (Trofanchuk et al. J Biol. Inorg. Chem. 2000, 5, 36-44). The g tensors were also calculated by density functional theory (DFT) methods using various structural models of the active site. The calculated g tensor of Ni-C is, concerning magnitudes and orientation, in good agreement with the experimental one for a formal Ni(III) oxidation state with a hydride (H-) bridge between the Ni and the Fe atom. Satisfying agreement is obtained for the Ni-L state when a formal Ni(l) oxidation state is assumed for this species with a proton (HI) removed from the bridge between the nickel and the iron atom.

The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 Angstrom resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H-2 in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 Angstrom and 160degrees, respectively, except for one case (1.72 Angstrom and 135degrees), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H-2-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm (similar to3000 M-1 cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (CO)-C-13-O-18). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography.