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Journal Article
Crystallization and preliminary X-ray diffraction analysis of two pH-dependent forms of a di-haem cytochrome c peroxidase from Pseudomonas nautica, Dias, João M., Bonifácio Cecília, Alves Teresa, Moura José J. G., Moura Isabel, and Romão Maria João , Acta Crystallographica Section D, Volume 58, Number 4, p.697-699, (2002) AbstractWebsite
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Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c, Samhan-Arias, A. K., Fortalezas S., Cordas C., Moura I., Moura J. J. G., and Gutierrez-Merino C. , Redox Biol, Volume 15, p.109-114, (2018)
Cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774. The relevance of the two calcium sites in the structure of the catalytic subunit (NrfA), Cunha, C. A., Macieira S., Dias J. M., Almeida G., Goncalves L. L., Costa C., Lampreia J., Huber R., Moura J. J., Moura I., and Romao M. J. , J Biol Chem, May 9, Volume 278, Number 19, p.17455-65, (2003) AbstractWebsite

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-A resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.

A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization, Alves, T., Besson S., Duarte L. C., Pettigrew G. W., Girio F. M. F., Devreese B., Vandenberghe I., Van Beeumen J., Fauque G., and Moura I. , Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology, Oct 12, Volume 1434, Number 2, p.248-259, (1999) AbstractWebsite

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 mu M and allowing a maximal catalytic centre activity of 116 000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature. (C) 1999 Elsevier Science B.V. All rights reserved.

A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization, Besson, S., Carneiro C., Moura J. J., Moura I., and Fauque G. , Anaerobe, Aug, Volume 1, Number 4, p.219-26, (1995) AbstractWebsite

Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments.

Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774, Liu, M. C., Costa C., Coutinho I. B., Moura J. J., Moura I., Xavier A. V., and Legall J. , J Bacteriol, Dec, Volume 170, Number 12, p.5545-51, (1988) AbstractWebsite

Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells.

Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation, Tiago, T., Aureliano M., and Moura J. J. , J Inorg Biochem, Nov, Volume 98, Number 11, p.1902-10, (2004) AbstractWebsite

Recently reported decameric vanadate (V(10)) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V(10) species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and decavanadate solutions, indicating that V(10) and tetrameric vanadate (V(4)) have the same binding sites in myosin S1. Concentrations as low as 50 muM decavanadate (5 muM V(10) species) induces 30% of protein cleavage, whereas 500 muM metavanadate is needed to attain the same extent of cleavage. After irradiation, V(10) species is rapidly decomposed, upon protein addition, forming vanadyl (V(4+)) species during the process. It was also observed by NMR line broadening experiments that, V(10) competes with V(4) for the myosin S1 binding sites, having a higher affinity. In addition, V(4) interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V(10) appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V(10).

Decavanadate interactions with actin: cysteine oxidation and vanadyl formation, Ramos, S., Duarte R. O., Moura J. J., and Aureliano M. , Dalton Trans, Oct 14, Number 38, p.7985-94, (2009) AbstractWebsite

Incubation of actin with decavanadate induces cysteine oxidation and oxidovanadium(IV) formation. The studies were performed combining kinetic with spectroscopic (NMR and EPR) methodologies. Although decavanadate is converted to labile oxovanadates, the rate of deoligomerization can be very slow (half-life time of 5.4 h, at 25 degrees C, with a first order kinetics), which effectively allows decavanadate to exist for some time under experimental conditions. It was observed that decavanadate inhibits F-actin-stimulated myosin ATPase activity with an IC(50) of 0.8 microM V(10) species, whereas 50 microM of vanadate or oxidovanadium(IV) only inhibits enzyme activity up to 25%. Moreover, from these three vanadium forms, only decavanadate induces the oxidation of the so called "fast" cysteines (or exposed cysteine, Cys-374) when the enzyme is in the polymerized and active form, F-actin, with an IC(50) of 1 microM V(10) species. Decavanadate exposition to F- and G-actin (monomeric form) promotes vanadate reduction since a typical EPR oxidovanadium(IV) spectrum was observed. Upon observation that V(10) reduces to oxidovanadium(IV), it is proposed that this cation interacts with G-actin (K(d) of 7.48 +/- 1.11 microM), and with F-actin (K(d) = 43.05 +/- 5.34 microM) with 1:1 and 4:1 stoichiometries, respectively, as observed by EPR upon protein titration with oxidovanadium(IV). The interaction of oxidovanadium(IV) with the protein may occur close to the ATP binding site of actin, eventually with lysine-336 and 3 water molecules.

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate, Ramos, S., Manuel M., Tiago T., Duarte R., Martins J., Gutierrez-Merino C., Moura J. J., and Aureliano M. , J Inorg Biochem, Nov, Volume 100, Number 11, p.1734-43, (2006) AbstractWebsite

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.

Design of Artificial Enzymes Using the Metals of the Periodic Table, J.J.G., Moura , Memories of the Class of Sciences, Academia das Ciências de Lisboa, (2020)
Designed Metal-ATCUN Derivatives: Redox and Non-redox-Based Applications Relevant for Chemistry, Biology, and Medicine, Maiti, B. K., Govil N., Kundu T., and J.J.G. Moura , iScience, Volume 23, p.101792, (2020)
Desulfoferrodoxin: a modular protein, Ascenso, C., Rusnak F., Cabrito I., Lima M. J., Naylor S., Moura I., and Moura J. J. , J Biol Inorg Chem, Dec, Volume 5, Number 6, p.720-9, (2000) AbstractWebsite

The gene encoding the non-heme iron-containing desulfoferrodoxin from Desulfovibrio vulgaris was cloned in two fragments in order to obtain polypeptides corresponding to the N- and C-terminal domains observed in the tertiary structure. These fragments were expressed in Escherichia coli, purified to homogeneity and biochemically and spectroscopically characterized. Both recombinant fragments behaved as independent metal-binding domains. The N-terminal fragment exhibited properties similar to desulforedoxin, as expected by the presence of a Fe(S-Cys)4 metal binding motif. The C-terminal fragment, which accommodates a Fe(Nepsilon-His)3(Ndelta-His)(S-Cys) center, was shown to have properties similar to neelaredoxin, except for the reaction with superoxide. The activities of desulfoferrodoxin and of the expressed C-terminal fragment were tested with superoxide in the presence and absence of cytochrome c. The results are consistent with superoxide reductase activity and a possible explanation for the low superoxide consumption in the superoxide dismutase activity assays is proposed.

Desulforedoxin: Preliminary X-ray diffraction study of a new iron-containing protein, Sieker, L. C., Jensen L. H., Bruschi M., Legall J., Moura I., and Xavier A. V. , Journal of Molecular Biology, Volume 144, Number 4, p.593-594, (1980) AbstractWebsite
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Desulforedoxin: proposed configuration and preliminary X-ray diffraction study of a two-iron two chain protein, Sieker, L. C., Bruschi M., Legall J., Moura I., and Xavier A. V. , Ciênc. Biol. (Portugal), Volume 5, p.145-147, (1980) Abstract
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Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe-4S] cluster, Rodrigues, P. M., Macedo A. L., Goodfellow B. J., Moura I., and Moura J. J. , J Biol Inorg Chem, Apr, Volume 11, Number 3, p.307-15, (2006) AbstractWebsite

Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe3S4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdIIint) with four 1H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdIIox and FdIIint were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe-4S] core in the oxidized state (DgFdIIox) was used here for the assignment of the same three ligands in the intermediate state (DgFdIIint). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm-1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdIIint. This study, together with the spin delocalization mechanism proposed here for DgFdIIint, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdIIox and DgFdIIint.

Desulfovibrio Gigas hydrogenase: redox properties of the nickel and iron-sulfur centers, Teixeira, M., Moura I., Xavier A. V., Dervartanian D. V., Legall J., Peck, H. D. Jr., Huynh B. H., and Moura J. J. , Eur J Biochem, Feb 15, Volume 130, Number 3, p.481-4, (1983) AbstractWebsite

Below 30 K, oxidized Desulfovibrio gigas hydrogenase presents an intense electron paramagnetic resonance (EPR) signal centered at g = 2.02, typical of an iron-sulfur center. In addition a rhombic EPR signal, attributed to Ni(III) species, is also observed [LeGall, J., Ljungdahl, P., Moura, I., Peck, H.D., Jr, Xavier, A.V., Moura, J.J.G., Teixeira, M., Huynh, B.H., and DerVartanian, D.V. (1982) Biochem. Biophys. Res. Commun. 106, 610-616; and Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E.C., (1982) FEBS Lett. 142, 289-292]. At higher temperatures (77 K) the iron-sulfur EPR signal is broader and all the EPR features of the rhombic nickel signal can easily be observed. We have now obtained additional information concerning the redox properties of these EPR active centers, using an EPR redox titration method in the presence of dye mediators at pH = 8.5. The mid-point potential was determined to be -70 mV for the Fe,S cluster and -220 mV for the Ni center. Intermediate oxidation states were obtained upon partial reduction with either dithionite or hydrogen. Although upon dithionite reduction the centers are reduced in the order of decreasing mid-point reduction potentials, under a hydrogen atmosphere the nickel center reduces preferentially. This suggests a catalytic involvement of the nickel redox center in the binding of hydrogen. Preliminary Mossbauer studies on Desulfovibrio gigas hydrogenase reveal the presence of a paramagnetic 3 Fe center and two 4 Fe centers. The 3 Fe center is responsible for the g = 2.02 EPR signal but the two 4 Fe centers have been so far undetectable by EPR.

Detection of nitric oxide by electron paramagnetic resonance spectroscopy: spin-trapping with iron-dithiocarbamates, Maia, L. B., and Moura J. J. G. , Methods Mol Biol, Volume 1424, p.81-102, (2016) Website
Determination of the active form of the tetranuclear copper sulfur cluster in nitrous oxide reductase, Johnston, E. M., Dell'Acqua S., Ramos S., Pauleta S. R., Moura I., and Solomon E. I. , J Am Chem Soc, Volume 136, p.614–617, (2014)
Development of a ferrocenyl-based MIP in supercritical carbon dioxide: Towards an electrochemical sensor for bisphenol A, Rebocho, S., Cordas C. M., Viveiros R., and Casimiro T. , J Supercrit Fluids, Volume 135, p.98-104, (2018) Website
Direct electrochemical reduction of carbon dioxide by a molybdenum-containing formate dehydrogenase, Cordas, C. M., Campaniço M., Baptista R., Maia L., Moura I., and Moura J. J. G. , J Inorg Biochem, Volume 196, p.110694, (2019) Website
Direct electrochemical study of the multiple redox centers of hydrogenase from Desulfovibrio gigas, Cordas, C. M., Moura I., and Moura J. J. , Bioelectrochemistry, Nov, Volume 74, Number 1, p.83-9, (2008) AbstractWebsite

Direct electrochemical response was first time observed for the redox centers of Desulfovibrio gigas [NiFe]-Hase, in non-turnover conditions, by cyclic voltammetry, in solution at glassy carbon electrode. The activation of the enzyme was achieved by reduction with H(2) and by electrochemical control and electrocatalytic activity was observed. The inactivation of the [NiFe]-Hase was also attained through potential control. All electrochemical data was obtained in the absence of enzyme inhibitors. The results are discussed in the context of the proposed mechanism currently accepted for activation/inactivation of [NiFe]-Hases.

Direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase, Correia dos Santos, M. M., Sousa P. M., Goncalves M. L., Romao M. J., Moura I., and Moura J. J. , Eur J Biochem, Apr, Volume 271, Number 7, p.1329-38, (2004) AbstractWebsite

This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.

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
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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.

Diverse biological roles of the tetrathiomolybdate anion, Maiti, B. K., and Moura J. J. G. , Coord Chem Rev, Volume 429, p.213635, (2020)