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Characterization of two dissimilatory sulfite reductases from sulfate-reducing bacteria, Huynh, B., Moura I., Lino A., Moura J., and Legall J. , Hyperfine Interactions, Volume 42, Number 1, p.905-908, (1988) AbstractWebsite

Mössbauer, EPR, and biochemical techniques were used to characterize two dissimilatory sulfite reductases: desulforubidin from Desulfovibrio baculatus strain DSM 1743 and desulfoviridin from Desulfovibrio gigas . For each molecule of desulforubidin, there are two sirohemes and four [4Fe−4S] clusters. The [4Fe−4S] clusters are in the diamagnetic 2+ oxidation state. The sirohemes are high-spin ferric (S=5/2) and each siroheme is exchanged-coupled to a [4Fe−4S] 2+ cluster. Such an exchange-coupled siroheme-[4Fe−4S] unit has also been found in the assimilatory sulfite reductase from Escherichia coli /1/ and in a low-molecular weight sulfite reductase from Desulfovibrio vulgaris /2/. For each molecule of defulfoviridin, there are two tetrahydroporphyrin groups and four [4Fe−4S] 2+ clusters. To our surprise, we discovered that about 80% of the tetrahydroporphyrin groups, however, do not bind iron.

Immunocytochemical localization of APS reductase and bisulfite reductase in three <i>Desulfovibrio</i> species, Kremer, D. R., Veenhuis M., Fauque G., Peck H. D., Legall J., Lampreia J., Moura J. J. G., and Hansen T. A. , Archives of Microbiology, Volume 150, Number 3, p.296-301, (1988) AbstractWebsite

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were embedded and ultrathin sections were incubated with antibodies and subsequently labeled with protein A-gold. The bisulfite reductase in all three strains and APS reductase in d. gigas and D. vulgaris were found in the cytoplasm. The labeling of d. thermophilus with APS reductase antibodies resulted in a distribution of gold particles over the cytoplasmic membrane region. The localization of the two enzymes is discussed with respect to the mechanism and energetics of dissimilatory sulfate reduction.

The molybdenum iron-sulphur protein from Desulfovibrio gigas as a form of aldehyde oxidase, Turner, N., Barata B., Bray R. C., Deistung J., Legall J., and Moura J. J. , Biochem J, May 1, Volume 243, Number 3, p.755-61, (1987) AbstractWebsite

The molybdenum iron-sulphur protein originally isolated from Desulfovibrio gigas by Moura, Xavier, Bruschi, Le Gall, Hall & Cammack [(1976) Biochem. Biophys. Res. Commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(V). The signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 HGz in 1H2O and 2H2O and computer simulations, to have parameters corresponding to those of the Slow signal from the inactive desulpho form of various molybdenum-containing hydroxylases. Another signal obtained on brief reduction of the protein with small amounts of dithionite was shown by e.p.r. difference techniques to be a Rapid type 2 signal, like that from the active form of such enzymes. In confirmation that the protein is a molybdenum-containing hydroxylase, activity measurements revealed that it had aldehyde:2,6-dichlorophenol-indophenol oxidoreductase activity. No such activity towards xanthine or purine was observed. Salicylaldehyde was a particularly good substrate, and treatment of the protein with it also gave rise to the Rapid signal. Molybdenum cofactor liberated from the protein was active in the nit-1 Neurospora crassa nitrate reductase assay. It is concluded that the protein is a form of an aldehyde oxidase or dehydrogenase. From the intensity of the e.p.r. signals and from enzyme activity measurements, 10-30% of the protein in the sample examined appeared to be in the functional form. The evolutionary significance of the protein, which may represent a primitive form of the enzyme rather than a degradation product, is discussed briefly.

On the active sites of the [NiFe] hydrogenase from Desulfovibrio gigas. Mossbauer and redox-titration studies, Huynh, B. H., Patil D. S., Moura I., Teixeira M., Moura J. J., Dervartanian D. V., Czechowski M. H., Prickril B. C., Peck, H. D. Jr., and Legall J. , J Biol Chem, Jan 15, Volume 262, Number 2, p.795-800, (1987) AbstractWebsite

The [NiFe] hydrogenase isolated from Desulfovibrio gigas was poised at different redox potentials and studied by Mossbauer spectroscopy. The data firmly establish that this hydrogenase contains four prosthetic groups: one nickel center, one [3Fe-xS], and two [4Fe-4S] clusters. In the native enzyme, both the nickel and the [3Fe-xS] cluster are EPR-active. At low temperature (4.2 K), the [3Fe-xS] cluster exhibits a paramagnetic Mossbauer spectrum typical for oxidized [3Fe-xS] clusters. At higher temperatures (greater than 20 K), the paramagnetic spectrum collapses into a quadrupole doublet with parameters magnitude of delta EQ magnitude of = 0.7 +/- 0.06 mm/s and delta = 0.36 +/- 0.06 mm/s, typical of high-spin Fe(III). The observed isomer shift is slightly larger than those observed for the three-iron clusters in D. gigas ferredoxin II (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) and in Azotobacter vinelandii ferredoxin I (Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796) and may indicate a different iron coordination environment. When D. gigas hydrogenase is poised at potentials lower than -80 mV (versus normal hydrogen electrode), the [3Fe-xS] cluster is reduced and becomes EPR-silent. The Mossbauer data indicate that the reduced [3Fe-xS] cluster remains intact, i.e. it does not interconvert into a [4Fe-4S] cluster. Also, the electronic properties of the reduced [3Fe-xS] cluster suggest that it is magnetically isolated from the other paramagnetic centers.

Characterization of the cytochrome system of a nitrogen-fixing strain of a sulfate-reducing bacterium: Desulfovibrio desulfuricans strain Berre-Eau, Moura, I., Fauque G., Legall J., Xavier A. V., and Moura J. J. , Eur J Biochem, Feb 2, Volume 162, Number 3, p.547-54, (1987) AbstractWebsite

Two c-type cytochromes were purified and characterized by electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopic techniques, from the sulfate-reducer nitrogen-fixing organism, Desulfovibrio desulfuricans strain Berre-Eau (NCIB 8387). The purification procedures included several chromatographic steps on alumina, carboxymethylcellulose and gel filtration. A tetrahaem and a monohaem cytochrome were identified. The multihaem cytochrome has visible, EPR and NMR spectra with general properties similar to other low-potential bis-histidinyl axially bound haem proteins, belonging to the class of tetrahaem cytochrome c3 isolated from other Desulfovibrio species. The monohaem cytochrome c553 is ascorbate-reducible and its EPR and NMR data are characteristic of a cytochrome with methionine-histidine ligation. Their properties are compared with other homologous proteins isolated from sulfate-reducing bacteria.

Characterization Of Electron-Transfer Proteins From The Nitrogen-Fixing Sulfate-Reducing Bacterium Desulfovibrio-Desulfuricans Berre-Eau, Fauque, G., Moura I., Xavier A. V., Galliano N., Moura J. J. G., and Legall J. , Biochemical Society Transactions, Dec, Volume 15, Number 6, p.1049-1050, (1987) AbstractWebsite
Nickel-[iron-sulfur]-selenium-containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic properties, Teixeira, M., Fauque G., Moura I., Lespinat P. A., Berlier Y., Prickril B., Peck, H. D. Jr., Xavier A. V., Legall J., and Moura J. J. , Eur J Biochem, Aug 17, Volume 167, Number 1, p.47-58, (1987) AbstractWebsite

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g approximately 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and approximately 2.0 (component I) and at 2.33, 2.24, and approximately 2.0 (component II). In the reduced state, after exposure to an H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers: center I (2.03, 1.89 and 1.86) detectable below 10 K, and center II (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 8942]. During the course of a redox titration at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Moessbauer study of D. gigas ferredoxin II and spin-coupling model for Fe3S4 cluster with valence delocalization, Papaefthymiou, V., Girerd J. J., Moura I., Moura J. J. G., and Muenck E. , Journal of the American Chemical Society, 1987/07/01, Volume 109, Number 15, p.4703-4710, (1987) AbstractWebsite
Evidence for the formation of a ZnFe3S4 cluster in Desulfovibrio gigas ferredoxin II, Surerus, Kristene K., Munck Eckard, Moura Isabel, Moura Jose J. G., and Legall Jean , Journal of the American Chemical Society, 1987/06/01, Volume 109, Number 12, p.3805-3807, (1987) AbstractWebsite
Proton NMR spectra of rubredoxins: new resonances assignable to .alpha.-CH and .beta.-CH2 hydrogens of cysteinate ligands to iron(II), Werth, Mark T., Kurtz Donald M., Moura Isabel, and Legall Jean , Journal of the American Chemical Society, 1987/01/01, Volume 109, Number 1, p.273-275, (1987) AbstractWebsite
Identification of three classes of hydrogenase in the genus, Desulfovibrio, Prickril, Benet C., He Shao-Hua, Li Ching, Menon Nanda, Choi Eui-Sung, Przybyla Alan E., DerVartanian Daniel V., Peck Jr Harry D., Fauque Guy, Legall Jean, Teixeira Miguel, Moura Isabel, Moura Jose J. G., Patil Daulat, and Huynh Boi H. , Biochemical and Biophysical Research Communications, Volume 149, Number 2, p.369-377, (1987) AbstractWebsite
Isolation and characterization of a rubredoxin and a flavodoxin from Desulfovibrio desulfuricans Berre-Eau, Fauque, Guy D., Moura Isabel, Moura José J. G., Xavier António V., Galliano Nicole, and Legall Jean , Febs Letters, Volume 215, Number 1, p.63-67, (1987) AbstractWebsite
Redox properties and activity studies on a nickel-containing hydrogenase isolated from a halophilic sulfate reducer Desulfovibrio salexigens, Teixeira, M., Moura I., Fauque G., Czechowski M., Berlier Y., Lespinat P. A., Legall J., Xavier A. V., and Moura J. J. , Biochimie, Jan, Volume 68, Number 1, p.75-84, (1986) AbstractWebsite

A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 mumoles H2 evolved/min/mg (an overall 180-fold purification with 20% recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12-15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A400/A280 = 0.275) and a molar absorbance of 54 mM-1cm-1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. Le Gall (1984), J. Mol. Cat., 23, 305-314). Upon longer incubation with H2 the "2.22" EPR signal decreases. During the course of a redox titration under H2, this EPR signal attains a maximal intensity around--380 mV. At redox states where this "2.22" signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g-values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the "2.22" signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with gmed approximately 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D2/H+ and H2 production measurements.

Low-spin sulfite reductases: a new homologous group of non-heme iron-siroheme proteins in anaerobic bacteria, Moura, I., Lino A. R., Moura J. J., Xavier A. V., Fauque G., Peck, H. D. Jr., and Legall J. , Biochem Biophys Res Commun, Dec 30, Volume 141, Number 3, p.1032-41, (1986) AbstractWebsite

Two new low molecular weight proteins with sulfite reductase activity, isolated from Methanosarcina barkeri (DSM 800) and Desulfuromonas acetoxidans (strain 5071), were studied by EPR and optical spectroscopic techniques. Both proteins have visible spectra similar to that of the low-spin sulfite reductase of Desulfovibrio vulgaris strain Hildenborough and no band at 715 nm, characteristic of high-spin Fe3+ complexes in isobacteriochlorins is observed. EPR shows that as isolated the siroheme is in a low-spin ferric state (S = 1/2) with g-values at 2.40, 2.30 and 1.88 for the Methanosarcina barkeri enzyme and g-values at 2.44, 2.33 and 1.81 for the Desulfuromonas acetoxidans enzyme. Chemical analysis shows that both proteins contain one siroheme and one [Fe4S4] center per polypeptidic chain. These results suggest that the low molecular weight, low-spin non-heme iron siroheme proteins represent a new homologous class of sulfite reductases common to anaerobic microorganisms.

Resonance Raman spectra of rubredoxin: new assignments and vibrational coupling mechanism from iron-54/iron-56 isotope shifts and variable-wavelength excitation, Czernuszewicz, Roman S., Legall Jean, Moura Isabel, and Spiro Thomas G. , Inorganic Chemistry, 1986/02/01, Volume 25, Number 5, p.696-700, (1986) AbstractWebsite
Evidence for the formation of a cobalt-iron-sulfur (CoFe3S4) cluster in Desulfovibrio gigas ferredoxin II, Moura, Isabel, Moura Jose J. G., Munck Eckard, Papaefthymiou Vasilios, and Legall Jean , Journal of the American Chemical Society, 1986/01/01, Volume 108, Number 2, p.349-351, (1986) AbstractWebsite
Purification and characterization of three proteins from a halophilic sulfate-reducing bacterium,<i>Desulfovibrio salexigens</i&gt, Czechowski, M., Fauque G., Galliano N., Dimon B., Moura I., Moura J. J. G., Xavier A. V., Barato B. A. S., Lino A. R., and Legall J. , Journal of Industrial Microbiology & Biotechnology, Volume 1, Number 3, p.139-147, (1986) AbstractWebsite
Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas, Teixeira, M., Moura I., Xavier A. V., Huynh B. H., Dervartanian D. V., Peck, H. D. Jr., Legall J., and Moura J. J. , J Biol Chem, Jul 25, Volume 260, Number 15, p.8942-50, (1985) AbstractWebsite

Desulfovibrio gigas hydrogenase (EC is a complex enzyme containing one nickel, one 3Fe, and two [Fe4S4] clusters (Teixeira, M., Moura, I., Xavier, A. V., Der Vartanian, D. V., LeGall, J., Peck, H. D., Jr., Huynh, B. H., and Moura, J. J. G. (1983) Eur. J. Biochem. 130, 481-484). This hydrogenase belongs to a class of enzymes that are inactive "as isolated" (the so-called "oxygen-stable hydrogenases") and must go through an activation process in order to express full activity. The state of characterization of the active centers of the enzyme as isolated prompted us to do a detailed analysis of the redox patterns, activation profile, and catalytic redox cycle of the enzyme in the presence of either the natural substrate (H2) or chemical reductants. The effect of natural cofactors, as cytochrome C3, was also studied. Special focus was given to the intermediate redox species generated during the catalytic cycle of the enzyme and to the midpoint redox potentials associated. The available information is discussed in terms of a "working hypothesis" for the mechanism of the [NiFe] hydrogenases from sulfate reducing organisms in the context of activation process and catalytic cycle.

Cobalt containing B12 cofactors from methanogenic bacteria - spectroscopic characterization, Lino, A. R., Xavier A. V., Moura I., Legall J., and Ljungdahl P. O. , Rev Portuguesa de Química, Volume 27, p.175-177, (1985) Abstract
Nickel X-ray absorption spectroscopy of Desulvovibrio gigas hydrogenase, Scott, R. A., Czechowski M., Dervartanian D. V., Legall J., Peck Jr H. D., and Moura I. , Rev Portuguesa de Química, Volume 27, p.67-70, (1985) Abstract
Nuclear-magnetic-resonance studies of Desulfuromonas acetoxidans cytochrome c551.5 (c7), Moura, J. G., Moore G. R., Williams R. J., Probst I., Legall J., and Xavier A. V. , Eur J Biochem, Nov 2, Volume 144, Number 3, p.433-40, (1984) AbstractWebsite

1H nuclear magnetic resonance (NMR) spectroscopy has been used to examine cytochrome c551.5 (c7) from the sulfur reducer, Desulfuromonas acetoxidans. This protein contains three hemes. Two stable oxidation states (the fully oxidized and the fully reduced) as well as intermediate oxidation states were studied. The axial ligands of the iron were found to be neutral histidines. The redox properties of cytochrome c7 were examined and good quantitative agreement found between the NMR results and previously reported redox potential measurements. The properties of cytochrome c7 are discussed together with those of the homologous tetraheme cytochromes c3 isolate from sulfate-reducing bacteria.

Interconversion from 3Fe into 4Fe clusters in the presence of Desulfovibrio gigas cell extracts, Moura, J. J., Legall J., and Xavier A. V. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.319-22, (1984) AbstractWebsite

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.

NMR and electron-paramagnetic-resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase), Villalain, J., Moura I., Liu M. C., Payne W. J., Legall J., Xavier A. V., and Moura J. J. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.305-12, (1984) AbstractWebsite

A dihaem cytochrome (Mr 37 400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11 607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous.

NMR studies of a dihaem cytochrome from Pseudomonas perfectomarinus (ATCC 14405), Moura, I., Liu M. C., Legall J., Peck, H. D. Jr., Payne W. J., Xavier A. V., and Moura J. J. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.297-303, (1984) AbstractWebsite

Pseudomonas perfectomarinus (ATCC 14405) dihaem cytochrome c552 was studied by 300-MHz proton magnetic resonance. Some of the haem resonances were assigned in the fully reduced and fully oxidized states. No evidence was found for methionine haem axial coordination. The oxidation-reduction equilibrium was studied in detail. Due to the large difference in mid-point redox potential between the two haems (+174 mV, for haem II and -180 mV for haem I) an intermediate oxidation state could be obtained containing reduced haem I and oxidized haem II. In this way the total paramagnetic shift at different oxidation levels could be decomposed in the intrinsic and extrinsic contributions. It was found that the two haems interact. The rate of electron exchange is slow on the NMR time scale. The redox equilibria are discussed for four possible redox species in solution.

NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c3, Santos, H., Moura J. J., Moura I., Legall J., and Xavier A. V. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.283-96, (1984) AbstractWebsite

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 NMR 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 NMR time scale (larger than 10(5) s-1). 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.