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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
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
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
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.

Discovery and characterization of a novel Dyp-type peroxidase from a marine actinobacterium isolated from Trondheim fjord, Norway, Cordas, C. M., Nguyen G. S., Valério G. V., Jønsson M., Sóllner K., Aune I., Wentzel A., and Moura J. J. G. , J Inorg Biochem, Volume 226, p.111651, (2022)
Dissimilatory Nitrate Reductase, Romão, M. J., Dias J. M., and Moura I. , Handbook of Metalloproteins , p.1075-1085, (2001) Abstract
Diverse biological roles of the tetrathiomolybdate anion, Maiti, B. K., and Moura J. J. G. , Coord Chem Rev, Volume 429, p.213635, (2020)
DNA damage and metal accumulation in four tissues of feral Octopus vulgaris from two coastal areas in Portugal, Raimundo, Joana, Costa Pedro M., Vale Carlos, Costa Maria Helena, and Moura Isabel , Ecotoxicology and Environmental Safety, Oct, Volume 73, Number 7, p.1543-1547, (2010) AbstractWebsite

The alkaline comet assay has been employed for the first time to estimate the basal DNA damage in the digestive gland, gills, kidney and gonads of Octopus vulgaris. Octopuses were captured in two coastal areas adjacent to the cities of Matosinhos (N) and Olhao (S), Portugal. The area of Matosinhos is influenced by discharges of the Douro River, city of Porto, industries and intensive agriculture, while Olhao is an important fisheries port. Previous works point to contrasting metal availability in the two coastal areas. Among the analysed tissues digestive gland presented the highest levels of Zn, Cu, Cd and Pb. Tissues of specimens from Matosinhos exhibited high levels of Cd and from Olhao enhanced Pb concentrations. The DNA damages in digestive gland, gills and kidney were more accentuated in specimens from Matosinhos than from Olhao, suggesting a stronger effect of contaminants. Elevated strand breakages were registered in digestive gland, recognised for its ability to store and detoxify accumulated metals. The DNA damages in kidney, gills and gonads were lower, reflecting reduced metal accumulation or efficient detoxification. The broad variability of damages in the three tissues may also mirror tissue function, specific defences to genotoxicants and cell-cycle turnover. (C) 2010 Elsevier Inc. All rights reserved.