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Electrochemical studies of the hexaheme nitrite reductase from Desulfovibrio desulfuricans ATCC 27774, Moreno, C., Costa C., Moura I., Legall J., Liu M. Y., Payne W. J., Van Dijk C., and Moura J. J. , Eur J Biochem, Feb 15, Volume 212, Number 1, p.79-86, (1993) AbstractWebsite

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.

Subunit composition, crystallization and preliminary crystallographic studies of the Desulfovibrio gigas aldehyde oxidoreductase containing molybdenum and [2Fe-2S] centers, Romao, M. J., Barata B. A., Archer M., Lobeck K., Moura I., Carrondo M. A., Legall J., Lottspeich F., Huber R., and Moura J. J. , Eur J Biochem, Aug 1, Volume 215, Number 3, p.729-32, (1993) AbstractWebsite

The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDS/PAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at 4 degrees C, pH 7.2, using isopropanol and MgCl2 as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6(1)22 or its enantiomorph, with cell dimensions a = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 x 10(-3) nm3/Da (2.5 A3/Da), consistent with the experimental crystal density, rho = 1.14 g/cm3. One dimer (approximately 2 x 100 kDa) is located on a crystallographic twofold axis.

Temperature-dependent proton NMR investigation of the electronic structure of the trinuclear iron cluster of the oxidized Desulfovibrio gigas ferredoxin II, Macedo, Anjos L., Moura Isabel, Moura Jose J. G., Legall Jean, and Huynh Boi Hanh , Inorganic Chemistry, 1993/03/01, Volume 32, Number 7, p.1101-1105, (1993) AbstractWebsite
Resonance Raman studies of nickel tetrathiolates and nickel-substituted rubredoxins and desulforedoxin, Huang, Yun Hua, Moura Isabel, Moura Jose J. G., Legall Jean, Park Jae Bum, Adams Michael W. W., and Johnson Michael K. , Inorganic Chemistry, 1993/02/01, Volume 32, Number 4, p.406-412, (1993) AbstractWebsite
Control of the spin state of the peroxidatic haem by calcium ions in cytochrome c peroxidase from Paracoccus denitrificans: A 1H NMR study, Prazeres, Susana, Moura Isabel, Moura José J. G., Gilmour Raymond, Goodhew Celia F., and Pettigrew Graham W. , Magnetic Resonance in Chemistry, Volume 31, Number 13, p.S68-S72, (1993) AbstractWebsite

Cytochrome c peroxidase from Paracoccus denitrificans LMD 52.44 was recently identified. The enzyme contains two c-type haems: one is reducible physiologically by cytochrome c550 from the same organism or non-physiologically by ascorbate (high-potential haem) and the other by dithionite (low-potential haem). The enzymatically active form of the peroxidase is the half-reduced enzyme state, in which the high-potential haem is in the iron(II) state and the low-potential haem is in the iron(III) state. It was found that the two haems interact and that the enzyme binds calcium ions near the haem sites which are necessary to promote its activation. In the oxidized form, the high-potential haem is in a high-spin and the low-potential haem is in a low-spin state. The half-reduction of the enzyme with ascorbate-diaminodurol changes the high-potential haem (high-spin) into a low-spin state and the low-potential haem converts from a low- into a high-spin state. This high-spin conversion of the low-potential haem is induced by the presence of calcium ions. These processes of reduction and spin state change can be easily resolved in time by removing the calcium from the enzyme using EDTA, facilitating the observation of the intermediate form by NMR.

Oxovanadium(IV) and amino acids—VI. The systems glycylglycine and glycylglycylglycine + VO2+; a potentiometric and spectroscopic study, Pessoa, Costa J., Luz S. M., Duarte R., Moura J. J. G., and Gillard R. D. , Polyhedron, Volume 12, Number 23, p.2857-2867, (1993) AbstractWebsite
Resonance Raman study of sirohydrochlorin and siroheme in sulfite reductases from sulfate reducing bacteria, Underwood-Lemons, Theresa, Moura Isabel, and To Yue Kwok , Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1157, Number 2, p.275-284, (1993) AbstractWebsite
Two-dimensional 1H NMR studies on Desulfovibrio gigas ferredoxins. Assignment of the iron-sulfur cluster cysteinyl ligand protons, Macedo, Anjos L., Palma Nuno P., Moura Isabel, Legall Jean, Wray Victor, and Moura José J. G. , Magnetic Resonance in Chemistry, Volume 31, Number 13, p.S59-S67, (1993) AbstractWebsite

1D and 2D 1H NMR studies are reported on the oxidized and reduced [4Fe-4S] cluster of Desulfovibrio gigas ferredoxin I (Fdl). Several low-field contact shifted resonances (fast relaxing) are assigned to β-CH2 and α-CH coordinated cysteinyl residues. NOESY patterns (supported by 1D NOE experiments) resolves four pairs of geminal β-CH2 protons at low-field. The cluster ligands are assigned non-specifically to Cys8, Cys11, Cys14 and Cys50, based on the X-ray structural analysis available for the oligomeric form, FdII, that contains a single [3Fe-4S] cluster. It was indicated in this case that Cys11 is not bound to the trinuclear cluster but is tilted towards the solvent. The presence of four pairs of geminal β-CH2 protons for FdI unambiguously proves the occupancy of the fourth site of the [3Fe-4S] complex and implies the coordination of the Cys11 at the cluster. Analysis of the oxidized form of FdII, using the same methodology as described for FdI, supports the presence of three cysteinyl ligands in the [3Fe-4S] core. Further, the combined use of the X-ray coordinates enables the specific assignment of the three cysteinyl ligands of the cluster, extending a previous assignment of Cys50. In addition, very broad resonances were detected for the reduced form of FdII in the low-field region around 200 ppm and in the high field region around −80 ppm.

The photochemical reaction between uranyl-nitrate and azulene, Burrows, H. D., Cardoso A. C., Formosinho S. J., Gil Ampc, Miguel M. D., Barata B., and Moura J. J. G. , Journal of Photochemistry and Photobiology a-Chemistry, Sep 30, Volume 68, Number 3, p.279-287, (1992) AbstractWebsite

On photolysis of solutions of azulene and uranyl nitrate in alcohols, a dark, amorphous precipitate is formed. Various analytical techniques show that this is a mixture of a uranium salt and an organic component, suggested to be polyazulene. The effects of various parameters on the yield of the product have been studied and it is found that oxygen facilitates the reaction. Electron spin resonance studies show that the product is paramagnetic, in agreement with the established ease of oxidation of polyazulene, and suggest that it is formed via electron transfer from azulene to excited uranyl ion, followed by successive dimerizations and deprotonations of radical cation intermediates.

Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium, Fauque, G., Czechowski M., Berlier Y. M., Lespinat P. A., Legall J., and Moura J. J. , Biochem Biophys Res Commun, May 15, Volume 184, Number 3, p.1256-60, (1992) AbstractWebsite

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774), Moura, I., Tavares P., Moura J. J., Ravi N., Huynh B. H., Liu M. Y., and Legall J. , J Biol Chem, Mar 5, Volume 267, Number 7, p.4489-96, (1992) AbstractWebsite

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.

Mossbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Spectral deconvolution of the heme components, Ravi, N., Moura I., Costa C., Teixeira M., Legall J., Moura J. J., and Huynh B. H. , Eur J Biochem, Mar 1, Volume 204, Number 2, p.779-82, (1992) AbstractWebsite

Mossbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mossbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic Mossbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic Mossbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.

Mossbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase, Barata, B. A., Liang J., Moura I., Legall J., Moura J. J., and Huynh B. H. , Eur J Biochem, Mar 1, Volume 204, Number 2, p.773-8, (1992) AbstractWebsite

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. Mossbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. Mossbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.

The nickel site in active Desulfovibrio baculatus [NiFeSe] hydrogenase is diamagnetic. Multifield saturation magnetization measurement of the spin state of Ni(II), Wang, C. P., Franco R., Moura J. J., Moura I., and Day E. P. , J Biol Chem, Apr 15, Volume 267, Number 11, p.7378-80, (1992) AbstractWebsite

The magnetic properties of the nickel(II) site in active Desulfovibrio baculatus (DSM 1743) [NiFeSe] hydrogenase have been measured using the multifield saturation magnetization technique. The periplasmic [NiFeSe] hydrogenase was isolated from bacteria grown in excess selenium in the presence of 57Fe. Saturation magnetization data were collected at three fixed fields (1.375, 2.75, 5.5 tesla) over the temperature range from 2 to 100 K. Mossbauer and EPR spectroscopies were used to characterize the magnetic state of the two [4Fe-4S] clusters of the enzyme and to quantitate the small amounts of iron impurities present in the sample. The nickel(II) site was found to be diamagnetic (low spin, S = 0). In combination with recent results from extended x-ray absorption fine structure studies, this magnetic state indicates that the nickel(II) site of active D. baculatus [NiFeSe] hydrogenase is five-coordinate.

NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans, Saraiva, Lígia M., Denariaz Gerard, Liu Ming- Y., Payne William J., Legall Jean, and Moura Isabel , European Journal of Biochemistry, Volume 204, Number 3, p.1131-1139, (1992) AbstractWebsite

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme–methionine coordination. The mid-point redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax∼ 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax∼ 3.5 converts mainly to a signal at g∼ 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.

The photochemical reaction between uranyl nitrate and azulene, Burrows, Hugh D., Cardoso Augusto C., Formosinho Sebastião J., Gil Ana M. P. C., da Miguel Maria Graça M., Barata Belamino, and J.G. Moura José , Journal of Photochemistry and Photobiology A: Chemistry, Volume 68, Number 3, p.279-287, (1992) AbstractWebsite
Spectroscopic studies on APS reductase isolated from the hyperthermophilic sulfate-reducing archaebacterium Archaeglobus fulgidus, Lampreia, J., Fauque G., Speich N., Dahl C., Moura I., Truper H. G., and Moura J. J. , Biochem Biophys Res Commun, Nov 27, Volume 181, Number 1, p.342-7, (1991) AbstractWebsite

Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP. A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304. Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme. The A. fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct [4Fe-4S] clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas. Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential.

Spectroscopic studies of cobalt and nickel substituted rubredoxin and desulforedoxin, Moura, I., Teixeira M., Legall J., and Moura J. J. , J Inorg Biochem, Nov, Volume 44, Number 2, p.127-39, (1991) AbstractWebsite

The single iron site of rubredoxin was replaced by nickel and cobalt. The near-infrared/visible/UV spectra of these metal derivatives show ligand-field transitions and charge-transfer bands which closely resemble those of simple tetrathiolate complexes, indicating a tetrahedral arrangement of the sulfur cysteinyl ligands around the metal core. The 1H NMR spectra of the nickel and cobalt derivatives reveal extremely low-field contact shifted resonances of one proton intensity assigned to beta-CH2 and alpha-CH cysteinyl protons. Other well resolved resonances shifted out of the main protein spectral envelope are also observed and probably arise from contact plus pseudocontact shift mechanisms. Rubredoxins from different sulfate reducers were metal substituted and assignments of aliphatic protons are tentatively proposed, taking advantage of the amino acid sequence homologies. The present data is promising in terms of structural analysis of the coordination sphere of the metal core. It was also shown that replacement of the iron atom of desulforedoxin, a close analogue of rubredoxin, by cobalt and nickel was possible.

Structural and functional approach toward a classification of the complex cytochrome c system found in sulfate-reducing bacteria, Moura, J. J., Costa C., Liu M. Y., Moura I., and Legall J. , Biochim Biophys Acta, May 23, Volume 1058, Number 1, p.61-6, (1991) AbstractWebsite

Following the discovery of the tetraheme cytochrome c3 in the strict anaerobic sulfate-reducing bacteria (Postgate, J.R. (1954) Biochem. J. 59, xi; Ishimoto et al. (1954) Bull. Chem. Soc. Japan 27, 564-565), a variety of c-type cytochromes (and others) have been reported, indicating that the array of heme proteins in these bacteria is complex. We are proposing here a tentative classification of sulfate- (and sulfur-) reducing bacteria cytochromes c based on: number of hemes per monomer, heme axial ligation, heme spin state and primary structures (whole or fragmentary). Different and complementary spectroscopic tools have been used to reveal the structural features of the heme sites.

Simulation of the electrochemical behavior of multi-redox systems. Current potential studies on multiheme cytochromes, Moreno, C., Campos A., Teixeira M., Legall J., Montenegro M. I., Moura I., Van Dijk C., and Moura J. G. , Eur J Biochem, Dec 5, Volume 202, Number 2, p.385-93, (1991) AbstractWebsite

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.

Information from e.p.r. spectroscopy on the iron-sulphur centres of the iron-molybdenum protein (aldehyde oxidoreductase) of Desulfovibrio gigas, Bray, R. C., Turner N. A., Legall J., Barata B. A., and Moura J. J. , Biochem J, Dec 15, Volume 280 ( Pt 3), p.817-20, (1991) AbstractWebsite

E.p.r. spectra of reduced iron-sulphur centres of the aldehyde oxidoreductase (iron-molybdenum protein) of Desulfovibrio gigas were recorded at X-band and Q-band frequencies and simulated. Results are consistent with the view that only two types of [2Fe-2S] clusters are present, as in eukaryotic molybdenum-containing hydroxylases. The data indicate the Fe/SI centre to be very similar, and the Fe/SII centre somewhat similar, to these centres in the eukaryotic enzymes.

Direct evidence of the metal-free nature of sirohydrochlorin in desulfoviridin, Lai, K. K., Moura Isabel, Liu Ming Y., Legall Jean, and To Yue Kwok , Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1060, Number 1, p.25-27, (1991) AbstractWebsite
The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies, Lampreia, J., Moura I., Teixeira M., Peck, H. D. Jr., Legall J., Huynh B. H., and Moura J. J. , Eur J Biochem, Mar 30, Volume 188, Number 3, p.653-64, (1990) AbstractWebsite

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). Mossbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mossbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.

Purification and characterization of desulfoferrodoxin. A novel protein from Desulfovibrio desulfuricans (ATCC 27774) and from Desulfovibrio vulgaris (strain Hildenborough) that contains a distorted rubredoxin center and a mononuclear ferrous center, Moura, I., Tavares P., Moura J. J., Ravi N., Huynh B. H., Liu M. Y., and Legall J. , J Biol Chem, Dec 15, Volume 265, Number 35, p.21596-602, (1990) AbstractWebsite

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. Mossbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.

Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli. A mass spectrometric study, Costa, C., Macedo A., Moura I., Moura J. J., Legall J., Berlier Y., Liu M. Y., and Payne W. J. , FEBS Lett, Dec 10, Volume 276, Number 1-2, p.67-70, (1990) AbstractWebsite

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.