The conversion of adrenaline to aminochromes by the human erythrocyte plasma membranes at pH 9.5 was shown to be a complex reaction that proceeded at least by two distinct phases. The first one, corresponding to the formation of adrenochrome, is catalyzed in the presence of the membranes, suggesting the involvement of an enzyme-mediated process. Active oxygen species were identified as intermediates during this phase. Oxygen radical scavengers (catalase and superoxide dismutase) suggested H2O2 and O2- involvement. Adrenochrome formation was stimulated by NADH indicating the participation of another enzyme (NADH dehydrogenase) which is known to be present in the human erythrocyte plasma membrane. The second phase, corresponding to the disappearance of adrenochrome, is also stimulated by NADH and inhibited in the presence of the membranes. In this reaction, adrenochrome is converted to aminochromes via adrenochrome semiquinone. The formation of radical species is demonstrated by EPR spectroscopy. The results led to the proposal of a mechanism for the formation of adrenochrome and other oxidation products from adrenaline.

The 275GHz electron-paramagnetic-resonance spectrometer we reported on in 2004 has been equipped with a new probe head, which contains a cavity especially designed for operation in continuous-wave mode. The sensitivity and signal stability that is achieved with this new probe head is illustrated with 275GHz continuous-wave spectra of a 1mM frozen solution of the complex Fe(III)-ethylenediamine tetra-acetic acid and of 10mM frozen solutions of the protein rubredoxin, which contains Fe(3+) in its active site, from three different organisms. The high quality of the spectra of the rubredoxins allows the determination of the zero-field-splitting parameters with an accuracy of 0.5GHz. The success of our approach results partially from the enhanced absolute sensitivity, which can be reached using a single-mode cavity. At least as important is the signal stability that we were able to achieve with the new probe head.

The diheme cytochrome c peroxidase from Paracoccus denitrificans was modified with the histidine-specific reagent diethyl pyrocarbonate. At low excess of reagent, 1 mol of histidine was modified in the oxidized enzyme, and modification was associated with loss of the ability to form the active state. With time, the modification reversed, and the ability to form the active state was recovered. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. However, the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity, and we propose a slow transfer of the ethoxyformyl group to an unidentified amino acid. The presence of CN- bound to the active peroxidatic site of the enzyme led to complete protection of the essential histidine from modification. Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (residues 251-338) showed that radioactivity was located in a peptide containing a single histidine at position 275. We propose that this conserved residue, in a highly conserved region, is central to the function of the active mixed-valence state.

The authors report the case of a 39-year-old male patient who had an ischemic stroke (complete infarction of right anterior cerebral circulation) and an acute myocardial infarction during the same year. Molecular study revealed he was homozygous for the 677C-->T mutation in the gene coding for methylenetetrahydrofolate reductase, a key enzyme of folate metabolism; deficiency of this enzyme is associated with increased cardiovascular risk and neurological lesions. Some considerations are put forward about hyperhomocysteinemia and the MTHFR 677C-->T mutation as cardiovascular risk factors.

The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement [Frazao et al. (1994) Acta Crystallogr. D50, 233-236] and refined at 1.75 A to an R-factor of 17.8%. When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept [heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues]. The three-dimensional structure of D. desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes [Palma et al. (1994) Biochemistry 33, 6394-6407]. This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling. The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations. Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure.

The aldehyde oxidoreductase from Desulfovibrio gigas belongs to the family of molybdenum hydroxylases. Besides a molybdenum cofactor which constitutes their active site, these enzymes contain two [2Fe-2S](2+,1+) clusters which are believed to transfer the electrons provided by the substrate to an acceptor which is either a FAD group or an electron-transferring protein. When the three metal centers of D. gigas AOR are simultaneously paramagnetic, splittings due to intercenter spin-spin interactions are visible when the EPR spectra are recorded at low temperatures. By studying quantitatively these interactions with a model based on the X-ray crystal structure, which takes into consideration the interactions between the magnetic moments carried by all the metal sites of the system, it is possible to determine the location of the reducible sites of the [2Fe-2S] clusters. When combined with the electron-transfer pathways proposed on the basis of the X-ray crystal structure, the results provide a detailed description of the electron-transfer system of D. gigas AOR.

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

The direct unmediated electrochemical response of the tetrahemic cytochrome c3 isolated from sulfate reducers Desulfovibrio baculatus (DSM 1743) and D. vulgaris (strain Hildenborough), was evaluated using different electrode systems [graphite (edge cut), gold, semiconductor (InO2) and mercury)] and different electrochemical methods (cyclic voltammetry and differential pulse voltammetry). A computer program was developed for the theoretical simulation of a complete cyclic voltammetry curve, based on the method proposed by Nicholson and Shain [Nicholson, R.S. & Shain, I. (1964) Anal. Chem. 36, 706-723], using the Gauss-Legendre method for calculation of the integral equations. The experimental data obtained for this multi-redox center protein was deconvoluted in to the four redox components using theoretically generated cyclic voltammetry curves and the four mid-point reduction potentials determined. The pH dependence of the four reduction potentials was evaluated using the deconvolution method described.

The same polypeptide chain (58 amino acids, 6 cysteines) is used to build up two ferredoxins in Desulfovibrio gigas a sulfate reducing organism. Ferredoxin II (FdII) contains a single [Fe3S4] core and ferredoxin I (FdI) mainly a [Fe4S4] core. The [Fe3S4] core can readily be interconverted into a [Fe4S4] complex (J.J.G. Moura, I. Moura, T.A. Kent, J.D. Lipscomb, B.H. Huynh, J. LeGall, A.V. Xavier, and E. Munck, J. Biol. Chem. 257, 6259 (1982)). This interconversion process suggested that the [Fe3S4] core could be used as a synthetic precursor for the formation of heterometal clusters. Co, Zn, Cd, and Ni derivatives were produced (I. Moura, J.J.G. Moura, E. Munck, V. Papaephthymiou, and J. LeGall, J. Am. Chem. Soc. 108, 349 (1986), K. Sureurs, E. Munck, I. Moura, J.J.G. Moura, and J. LeGall, J. Am. Chem. Soc. 109, 3805 (1986), and A.L. Macedo, I. Moura, J.J.G. Moura, K. Surerus, and E. Munck, unpublished results). The redox properties of a series of heterometal clusters (MFe3S4] are assessed using direct electrochemistry (square wave voltammetry--SWV) promoted by Mg(II) at a glassy carbon electrode (derivatives: Cd (-495 mV), Fe (-420 mV), Ni (-360 mV), and Co (-245 mV) vs normal hydrogen electrode (NHE)). In parallel, the electrochemical behavior (cyclic voltammetry--CV, differential pulse voltammetry--DPV and SWV) of FdI and FdII were investigated as well as the cluster interconversion process. In addition to the +1/0 (3Fe cluster) and +2/+1 (4Fe cluster) redox transitions, a very negative redox step, at -690 mV, was detected for the 3Fe core, reminiscent of a postulated further 2e- reduction step, as proposed for D. africanus ferredoxin III by F.A. Armstrong, S.J. George, R. Cammack, E.C. Hatchikian, and A.J. Thomson, Biochem. J. 264, 265 (1989). The electrochemical redox potential values are compared with those determined by independent methods (namely by electron paramagnetic resonance (EPR) and visible spectroscopy).

The kinetics of electron transfer between the Desulfovibrio gigas hydrogenase and several electron-transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, square-wave voltammetry and chronoamperometry. The cytochrome c3 from Desulfovibrio vulgaris (Hildenborough), Desulfovibrio desulfuricans (Norway 4), Desulfovibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron-transfer proteins such as ferredoxin I, ferredoxin II and rubredoxin from D. gigas were tentatively used as redox carriers. Only ferredoxin II was effective in mediating electron exchange between hydrogenase and the working electrode. The second-order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mechanism [Moreno, C., Costa, C., Moura, I., Le Gall, J., Liu, M. Y., Payne, W. J., van Dijk, C. & Moura, J. J. G. (1993) Eur. J. Biochem. 212, 79-86] and the results obtained by cyclic voltammetry were compared with those obtained by chronoamperometry. Values for k of 10(5)-10(6) M-1 s-1 (cytochrome c3 as electron carrier) and 10(4) M-1 s-1 (ferredoxin II as the electron carrier) were determined. The rate-constant values are discussed in terms of the existence of an electrostatic interaction between the electrode surface and the redox carrier and between the redox carrier and a positively charged part of the enzyme.

The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 10(6)-10(8) M-1 s-1 and increase in the direction D. desulfuricans cytochrome c3-->D. vulgaris cytochrome c3-->methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved. Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility). Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.

Formate dehydrogenases (FDHs) are enzymes that catalyze the formate oxidation to carbon dioxide and that contain either Mo or W in a mononuclear form in the active site. In the present work, the influence of Mo and W salts on the production of FDH by Desulfovibrio alaskensis NCIMB 13491 was studied. Two different FDHs, one containing W (W-FDH) and a second incorporating either Mo or W (Mo/W-FDH), were purified. Both enzymes were isolated from cells grown in a medium supplemented with 1 muM molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 muM tungstate. We demonstrated that the genes encoding the Mo/W-FDH are strongly downregulated by W and slightly upregulated by Mo. Metal effects on the expression level of the genes encoding the W-FDH were less significant. Furthermore, the expression levels of the genes encoding proteins involved in molybdate and tungstate transport are downregulated under the experimental conditions evaluated in this work. The molecular and biochemical properties of these enzymes and the selective incorporation of either Mo or W are discussed.

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. Mossbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic Mossbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.

Desulfovibrio gigas ferredoxin II (FdII) contains a single 3Fe cluster [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V., and Munck, E. (1980) J. Biol. Chem. 255, 3242-3244]. In the oxidized state the protein exhibits an intense electron paramagnetic resonance (EPR) signal at g = 2.02. Upon one-electron reduction the center becomes EPR silent. In the presence of D. gigas crude cell extracts, devoid of acidic electron carriers and supplemented with pyruvate and FdII, an EPR signal typical of reduced [4Fe-4S] centers is obtained. The appearance of this signal correlates with the beginning of stimulation of the phosphoroclastic reaction as judged by the production of H2. These results, supported by the occurrence of easy chemical conversion of the 3Fe cluster of D. gigas ferredoxin into 4Fe structures [Moura, J.J.G., Moura, I., Kent, T.A., Lipscomb, J.D., Huynh, B.H., LeGall, J., Xavier, A.V., and Munch, E. (1982) J. Biol. Chem. 257, 6259-6267], suggest that cluster conversion takes place in conditions close to the situation in vivo. This cluster interconversion is discussed in the context of some of the relevant metabolic pathways of Desulfovibrio spp.

The most thoroughly characterized non-heme iron center in biology is Rubredoxin, the simplest member of the iron-sulfur: class of metalloproteins. Rubredoxin contains a high-spin iron atom with tetrahedral coordination by four cysteinyl sulfur atoms. A structural variant of this center is found in Desulforedoxin, the smallest known Rubredoxin type protein. The 3D structure of both Rd and Dr has been determined at high resolution. These two proteins can therefore be used as case studies in which structural control by the polypeptide chain over the metal site can be discussed in detail.