The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

The three-dimensional crystal structure of the copper-containing nitrite reductase (NIR) from Achromobacter cycloclastes has been determined to 2.3 angstrom (A) resolution by isomorphous replacement. The monomer has two Greek key beta-barrel domains similar to that of plastocyanin and contains two copper sites. The enzyme is a trimer both in the crystal and in solution. The two copper atoms in the monomer comprise one type I copper site (Cu-I; two His, one Cys, and one Met ligands) and one putative type II copper site (Cu-II; three His and one solvent ligands). Although ligated by adjacent amino acids Cu-I and Cu-II are approximately 12.5 A apart. Cu-II is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the trimer. The Cu-II site is at the bottom of a 12 A deep solvent channel and is the site to which the substrate (NO2-) binds, as evidenced by difference density maps of substrate-soaked and native crystals.

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxidoreductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at

The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc,) are rationalised by a model which involves both homotropic (e-/e-) and heterotropic (e-/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the DVC, have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.Keywords: cooperativity ; energy transduction ; multiheme cytochrome ; NMR ; redox-Bohr.The functional cooperativity between different regions of some proteins [ l ] is a fundamental property to control and coordinate important chemical events in the living cell. Although the molecular basis for the fine regulation of several types of cooperativity mechanisms has been successfully established [2], little is known about the structural basis for electron/electron and electron/proton cooperativities and their role either in electron transfer or in energy transduction [3].Desulfovibrio spp. cytochrome c, is a small (=14 m a ) , monomeric tetrahaem protein which exhibits cooperativity between the four haems and acidmase group(s): the haem redox potentials are pH dependent (redox-Bohr effect) and each haem redox potential is dependent on the oxidation state of the other three haems (redox interaction potentials) [4-61. Due to its small size and the fact that the haems are diamagnetic in the reduced state and paramagnetic in the oxidised one, NMR is particularly well suited to characterise this protein from the structural and thermodynamic point of view [4,[7][8][9][10][11][12][13][14][15].Furthermore, several X-ray structures are available for cytochromes c3 from Desulfovibrio spp. [16-231.The thermodynamic properties of cytochrome c, have been analysed by previous NMR studies [4,5,8,15, 241. In the first of these studies an NMR data set obtained at two discrete pH values for Desulfovibrio gigas cytochrome c, was used to calculate nine parameters (three relative microscopic redox potentials and six haem-haem redox interactions) for each pH value, independently treated. The redox interaction potentials were fixed according to the maximum concentration reached by the intermediate oxidation stages (defined according to the number of oxidised haems) in redox titrations followed by NMR [4]. Using the same NMR data set, a second study proposed a model with 21 parameters in which the four haem redox potentials ...

The first crystal structure of a native di-iron center in an iron-storage protein (bacterio)ferritin is reported. The protein, isolated from the anaerobic bacterium Desulfovibrio desulfuricans, has the unique property of having Fe-coproporphyrin III as its heme cofactor. The three-dimensional structure of this bacterioferritin was determined in three distinct catalytic/redox states by X-ray crystallography (at 1.95, 2.05 and 2.35 A resolution), corresponding to different intermediates of the di-iron ferroxidase site. Conformational changes associated with these intermediates support the idea of a route for iron entry into the protein shell through a pore that passes through the di-iron center. Molecular surface and electrostatic potential calculations also suggest the presence of another ion channel, distant from the channels at the three- and four-fold axes proposed as points of entry for the iron atoms.

The different nutritional properties of several Desulfovibrio desulfuricans strains suggest that either the strains are misclassified or there is a high degree of phenotypic diversity within the genus Desulfovibrio. The obligately anaerobic, dissimilatory, sulfate-reducing bacteria utilize sulfate as their terminal electron acceptor and derive their energy for growth from the oxidation of organic compounds and hydrogen gas (27). Among the genera of sulfate-reducing bacteria, perhaps the most thoroughly studied species are classified in the genus Desulfovibrio. The Desulfovibrio species which have been characterized are nutritionally similar; they are readily enriched with lactate or hydrogen and utilize only a few other substrates (38). Diagnostically, Desulfovibrio species test positive (with some exceptions) for the sulfite reductase desulfoviridin and contain the unique tetraheme cytochrome C3 (27). These observations have contributed to the notion that although the sulfate-reducing bacteria are ecologically very significant, they comprise a small and nutritionally limited group. However, recent isolations and descriptions of new genera have dispelled this view (38). At present, more than 10 genera of sulfate-reducing eubacteria (one of which is affiliated with gram-positive bacteria) and an extremely thermophilic sulfate-reducing archaebacterium have been described (36, 39).While the nutritional versatility and phylogenetic diversity of the sulfate-reducing bacteria have been established, questions of relatedness among species of the genus Desulfovibrio remain unanswered. Postgate has stated that the "taxonomic picture" of the genus is "imperfect" (27

The proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio gigas were examined while varying the pH and the redox potential. The analysis of the N M R reoxidation pattern was based on a model for the electron distribution between the four haems that takes into account haem-haem redox interactions. The intramolecular electron exchange is fast on the N M R time scale (larger than lo5 s-I ) . The NMR data concerning the pH dependence of the chemical shift of haem methyl resonances in different oxidation steps and resonance intensities are not compatible with a non-interacting model and can be explained assuming a redox interaction between the haems. A complete analysis at pH* = 7.2 and 9.6, shows that the haem-haem interacting potentials cover a range from -50 mV to +60 mV. The midpoint redox potentials of some of the haems, as well as some of their interacting potentials, are pH-dependent. The physiological relevance of the modulation of the haem midpoint redox potentials by both the pH and the redox potential of the solution is discussed.Cy tochromc c3 ( M , 13 000) is a multihaem protein found in anaerobic sulfate-reducing bacteria belonging to the genus Desulfuvibrin. Each molecule contains four haems with an unusually low redox potential. They are covalently attached to the polypeptide chain by thioether linkages provided by cysteinyl residues and two histidines are used as axial ligands.Cytochrome [39] have been applied to elucidate the mechanism of electron transfer in cytochrome c3.The midpoint redox potentials of the four haems are different, in general. EPR measurements coupled with potentiometric titrations were performed to determine the midpoint redox potentials of the individual haems of D. gigas cytochrome c3 [27] (-235mV, -235mV, -306mV, -315mV) [35]; the experiments were fitted by digital simulations and the best fit was obtained with -220mV, -272mV, -292mV, and --310mV, for the four macroscopic midpoint redox potentials. Bianco et al. [36] reported values of -170mV, -310mV, -360mV and -400mV for the half-wave potentials of cytochrome c3 from D. desidfuricans (Norway 4). However. it should be emphasized that the individual potentials obtained from electrochemical studies are macroscopic rather than microscopic parameters and can not be compared directly with the values obtained from EPR measurements. Furthermore, thc EPR measurements

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