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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.

One Electron Reduced Square Planar Bis(benzene-1,2-dithiolato) Copper Dianionic Complex and Redox Switch by O2/HO-, Maiti, B. K., Maia L. B., Pal K., Pakira B., Aviles T., Moura I., Pauleta S. R., Nuñez J. L., Rizzi A. C., Brondino C. D., Sarkar S., and Moura J. J. G. , Inorg Chem, Volume 53, p.12799-12808, (2014)
Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis, Carepo, M. S., Carreira C., Grazina R., Zakrzewska M. E., Dolla A., Aubert C., Pauleta S. R., Moura J. J. G., and Moura I. , J Biol Inorg Chem, Volume 21, p.53-62, (2016)
Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin, Auchere, F., Sikkink R., Cordas C., Raleiras P., Tavares P., Moura I., and Moura J. J. , J Biol Inorg Chem, Oct, Volume 9, Number 7, p.839-49, (2004) AbstractWebsite

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy, Xavier, A. V., Moura J. J., Legall J., and Dervartanian D. V. , Biochimie, Volume 61, Number 5-6, p.689-95, (1979) AbstractWebsite

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.

Oxidation-reduction studies of the Mo-(2Fe-2S) protein from Desulfovibrio gigas, Moura, J. J., Xavier A. V., Cammack R., Hall D. O., Bruschi M., and Legall J. , Biochem J, Aug 1, Volume 173, Number 2, p.419-25, (1978) AbstractWebsite

Potentiometric titration followed by e.p.r. measurements were used to determine the midpoint reduction potentials of the redox centres of a molybdenum-containing iron-sulphur protein previously isolated from Desulfovibrio gigas, a sulphate-reducing bacterium (Moura, Xavier, Bruschi, Le Gall, Hall & Cammack (1976) Biochem. Biophys. Res. Commun. 728 782-789; Moura, Xavier, Bruschi, Le Gall & Cabral (1977) J. Less Common Metals 54, 555-562). The iron-sulphur centres could readily be distinguished into three types by means of g values, temperature effect, oxidation-reduction potential values and reduction rates. The type-I Fe-S centres are observed at 77 K. They show mid-point potential values of -260mV (Fe-S type IA) and -440 mV (Fe-S type IB). Centres of types IA and IB appear to have similar spectra at 77 K and 24 K. The Fe-S type-II centres are only observed below 65 K and have a midpoint potential of -28mV. Long equilibration times (30 min) with dye mediators under reducing conditions were necessary to observe the very slow equilibrating molybdenum signals. The potential values associated with this signal were estimated to be approx. -415 mV for Mo(VI)/Mo(V) and-530mV for Mo(V)/Mo(IV).

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
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Oxovanadium(IV) complexes of the dipeptides glycyl-L-aspartic acid, L-aspartylglycine and related ligands; a spectroscopic and potentiometric study, Pessoa, J. C., Gajda T., Gillard R. D., Kiss T., Luz S. M., Moura J. J. G., Tomaz I., Telo J. P., and Torok I. , Journal of the Chemical Society-Dalton Transactions, Nov 7, Number 21, p.3587-3600, (1998) AbstractWebsite

The equilibria in the systems VO2+ + L (L = Gly-L-Asp, L-Asp-Gly, N-acetyl-L-aspartic acid or succinic acid) have been studied at 25 degrees C and 0.2 mol dm(3) K(CI) medium by a combination of potentiometric and spectroscopic methods (ESR, circular dichroism and visible absorption). Formation constants were calculated from pH-metric data with total oxovanadium(Iv) concentrations of(0.6-4) x 10(-3) mol dm(-3) and ligand-to-metal (L:M) ratios of 2-8 (AspGly) or 4-15: 1 (other systems). The position of the Asp residue in the peptide chain affects the co-ordination mode of the ligands: while in the GlyAsp system bis complexes start to form at pH less than 2, for AspGly only 1 : 1 complexes form, with relatively high CD signal. The co-ordination behaviour of N-acetyl-L-aspartic and succinic acids is similar. The results of potentiometric and spectroscopic methods are self consistent. Isomeric structures are discussed for each stoichiometry proposed and the results compared with those for L-aspartic acid and dipeptides with non-coordinating side chains.

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Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase, Pauleta, S. R., Guerlesquin F., Goodhew C. F., Devreese B., Van Beeumen J., Pereira A. S., Moura I., and Pettigrew G. W. , Biochemistry, Sep 7, Volume 43, Number 35, p.11214-11225, (2004) AbstractWebsite

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

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.

Periplasmic nitrate reductase and formate dehydrogenase: similar molecular architectures with very different enzymatic activities, Cerqueira, N., Gonzalez P. J., Fernandes P. A., Moura J. J. G., and Ramos M. J. , Acc Chem Res, Volume 48, p.2875−2884, (2015)
Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum, Najmudin, S., Gonzalez P. J., Trincao J., Coelho C., Mukhopadhyay A., Cerqueira N. M., Romao C. C., Moura I., Moura J. J., Brondino C. D., and Romao M. J. , J Biol Inorg Chem, Jun, Volume 13, Number 5, p.737-53, (2008) AbstractWebsite

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

Periplasmic nitrate reductases and formate dehydrogenases: Biological control of the chemical properties of Mo and W for fine tuning of reactivity, substrate specificity and metabolic role, Gonzalez, P. J., Rivas M. G., Mota C. S., Brondino C. D., Moura I., and Moura J. J. G. , Coord Chem Rev, Volume 257, p.315-331, (2013)
Peroxidase-like activity of cytochrome b5 is triggered upon hemichrome formation in alkaline pH, Samhan-Arias, A., Maia L. B., Cordas C. M., Moura I., Gutierrez-Merino C., and Moura J. J. G. , BBA - Proteins and Proteomics, Volume 1866, p.373-378, (2018)
Perturbation of membrane dynamics in nerve cells as an early event during bilirubin-induced apoptosis, Rodrigues, C. M., Sola S., Castro R. E., Laires P. A., Brites D., and Moura J. J. , J Lipid Res, Jun, Volume 43, Number 6, p.885-94, (2002) AbstractWebsite

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

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
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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.

Physico-chemical and Spectroscopic Properties of the Monohemic Cytochrome C552 from Pseudomonas nautica 617, Saraiva, Lígia M., Fauque Guy, Besson Stéphane, and Moura Isabel , European Journal of Biochemistry, Volume 224, Number 3, p.1011-1017, (1994) AbstractWebsite

A c-type monohemic ferricytochrome c552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome c552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome c552 is typical of a low-spin ferric heme.

Potential therapeutic approaches for a sleeping pathogen: tuberculosis a case for bioinorganic chemistry, Sousa, E. H. S., Diógenes I. C. N., Lopes L. G. F., and Moura J. J. G. , J Biol Inorg Chem, Volume 25, p.685, (2020)
Predicting Protein-Protein Interactions Using BiGGER: Case Studies, Almeida, R. M., Dell'Acqua S., Krippahl L., Moura J. J. G., and Pauleta S. R. , Molecules, Volume 21, p.1037, (2016) Website
Prediction of Signal Peptides and Signal Anchors of Cytocrome c Nitrite Reductase from Desulfovibrio desulfuricans ATCC 27774 Using Bioinformatic Tools, Gonçalves, L. L., Almeida M. G., Lampreia J., Moura J. J. G., and Moura I. , Essays in Bioinformatics, Volume Vol. 368, p.203-208, (2005) Abstract

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Preliminary crystallographic analysis and further characterization of a dodecaheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774, Coelho, A. V., Matias P. M., Sieker L. C., Morais J., Carrondo M. A., Lampreia J., Costa C., Moura J. J., Moura I., and Legall J. , Acta Crystallogr D Biol Crystallogr, Nov 1, Volume 52, Number Pt 6, p.1202-8, (1996) AbstractWebsite

Dodecaheme cytochrome c has been purified from Desulfovibrio (D.) desulfuricans ATCC 27774 cells grown under both nitrate and sulfate-respiring conditions. Therefore, it is likely to play a role in the electron-transfer system of both respiratory chains. Its molecular mass (37768 kDa) was determined by electrospray mass spectrometry. Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the cytochromes of the c(3) type from sulfate-reducing bacteria sequenced at present. The midpoint redox potentials of this cytochrome were estimated to be -68, -120, -248 and -310 mV. Electron paramagnetic resonance spectroscopy of the oxidized cytochrome shows several low-spin components with a g(max) spreading from 3.254 to 2.983. Two crystalline forms were obtained by vapour diffusion from a solution containing 2% PEG 6000 and 0.25-0.75 M acetate buffer pH = 5.5. Both crystals belong to monoclinic space groups: one is P2(1), with a = 61.00, b = 106.19, c = 82.05 A, beta = 103.61 degrees, and the other is C2 with a = 152.17, b = 98.45, c = 89.24 A, beta = 119.18 degrees. Density measurements of the P2(1) crystals suggest that there are two independent molecules in the asymmetric unit. Self-rotation function calculations indicate, in both crystal forms, the presence of a non-crystallographic axis perpendicular to the crystallographic twofold axis. This result and the calculated values for the volume per unit molecular weight of the C2 crystals suggest the presence of two or four molecules in the asymmetric unit.

Preliminary crystallographic analysis of the oxidized form of a two mono-nuclear iron centres protein from Desulfovibrio desulfuricans ATCC 27774, Coelho, A. V., Matias P. M., Carrondo M. A., Tavares P., Moura J. J., Moura I., Fulop V., Hajdu J., and Legall J. , Protein Sci, Jun, Volume 5, Number 6, p.1189-91, (1996) AbstractWebsite

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A, b = 80.9 A, c = 53.9 A, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas, Legall, J., Ljungdahl P. O., Moura I., Peck, H. D. Jr., Xavier A. V., Moura J. J., Teixera M., Huynh B. H., and Dervartanian D. V. , Biochem Biophys Res Commun, May 31, Volume 106, Number 2, p.610-6, (1982) AbstractWebsite
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Primary sequence, oxidation-reduction potentials and tertiary-structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin, Caldeira, J., Palma P. N., Regalla M., Lampreia J., Calvete J., Schafer W., Legall J., Moura I., and Moura J. J. , Eur J Biochem, Mar 15, Volume 220, Number 3, p.987-95, (1994) AbstractWebsite

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate-reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, -387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J.J.G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K.D., Sieker, L.C., Jensen, L.H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.