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NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production, Maia, L., Duarte R. O., Ponces-Freire A., Moura J. J., and Mira L. , J Biol Inorg Chem, Aug, Volume 12, Number 6, p.777-87, (2007) AbstractWebsite

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.

Native Protein Template Assisted Synthesis of Non-Native Metal-Sulfur Clusters, B.K., Maiti, and J.J.G. Moura , BioChem, Volume 2, p.182-197, (2022)
A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase, Almeida, M. G., Silveira C. M., Guigliarelli B., Bertrand P., Moura J. J., Moura I., and Leger C. , FEBS Lett, Jan 23, Volume 581, Number 2, p.284-8, (2007) AbstractWebsite

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.

Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase, Jovanovic, T., Ascenso C., Hazlett K. R., Sikkink R., Krebs C., Litwiller R., Benson L. M., Moura I., Moura J. J., Radolf J. D., Huynh B. H., Naylor S., and Rusnak F. , J Biol Chem, Sep 15, Volume 275, Number 37, p.28439-48, (2000) AbstractWebsite

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mossbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

A new CuZ active form in the catalytic reduction of N(2)O by nitrous oxide reductase from Pseudomonas nautica, Dell'Acqua, S., Pauleta S. R., Paes de Sousa P. M., Monzani E., Casella L., Moura J. J., and Moura I. , J Biol Inorg Chem, Aug, Volume 15, Number 6, p.967-76, (2010) AbstractWebsite

The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.

New findings for in-gel digestion accelerated by high-intensity focused ultrasound for protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Carreira, R. J., Cordeiro F. M., Moro A. J., Rivas M. G., Rial-Otero R., Gaspar E. M., Moura I., and Capelo J. L. , Journal of Chromatography A, Jun 15, Volume 1153, Number 1-2, p.291-299, (2007) AbstractWebsite

New findings in sample treatment based on high-intensity focused ultrasound (HIFU) for protein digestion after polyacrylamide gel electrophoresis separation are presented. The following variables were studied: (i) sample volume; (ii) sonotrode diameter; (iii) previous protein denaturation; (iv) cooling; (v) enzyme concentration; and (vi) protein concentration. Results showed that positive protein identification could be done after protein separation by gel electrophoresis through peptide mass fingerprint (PMF) in a volume as low as 25 mu L. The time needed was less than 2 min and no cooling was necessary. The importance of the sonotrode diameter was negligible. On the other hand, protein denaturation before sonication was a trade-off for the success of procedure here described. The protein coverage was raised from 5 to 30%, and the number of peptides matching the proteins was also increased in a percentage ranging 10-100% when the classical overnight treatment is compared with the proposed HIFU procedure. The minimum amount of protein that can be identified using the HIFU sample treatment by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 0.06 mu g. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 mu g/mL. (c) 2006 Elsevier B.V. All rights reserved.

New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway, Folgosa, F., Cordas C. M., Santos J. A., Pereira A. S., Moura J. J., Tavares P., and Moura I. , Biochem J, Sep 15, Volume 438, Number 3, p.485-94, (2011) AbstractWebsite

SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 x 10 M(1) . s(1) and 1.3 x 10 M(1) . s(1) for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 x 10 M(1) . s(1) for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.

A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774, Gavel, O. Y., Bursakov S. A., Di Rocco G., Trincao J., Pickering I. J., George G. N., Calvete J. J., Shnyrov V. L., Brondino C. D., Pereira A. S., Lampreia J., Tavares P., Moura J. J., and Moura I. , J Inorg Biochem, May-Jun, Volume 102, Number 5-6, p.1380-95, (2008) AbstractWebsite

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

Nickel - a redox catalytic site in hydrogenase, Moura, J. J. G., Teixeira M., Moura I., Xavier A. V., and Legall J. , Journal of Molecular Catalysis, Volume 23, Number 2–3, p.303-314, (1984) AbstractWebsite
Nickel containing hydrogenases, Xavier, A. V., Teixeira M., Moura I., Moura J. J. G., and Legall J. , Inorganica Chimica Acta, Volume 79, p.13-14, (1983) AbstractWebsite
The nickel site in active Desulfovibrio baculatus [NiFeSe] hydrogenase is diamagnetic. Multifield saturation magnetization measurement of the spin state of Ni(II), Wang, C. P., Franco R., Moura J. J., Moura I., and Day E. P. , J Biol Chem, Apr 15, Volume 267, Number 11, p.7378-80, (1992) AbstractWebsite

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

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
NICKEL-CONTAINING HYDROGENASES, Moura, J. J. G., Moura I., Teixeira M., Xavier A. V., Fauque G. D., and Legall J. , Metal Ions in Biological Systems, 1988, Volume 23, p.285-314, (1988) 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)

NiII -ATCUN-catalyzed tyrosine nitration in the presence of nitrite and sulfite, Maiti, B. K., Maia L. B., Moura I., and Moura J. J. G. , Chem Eur J, Volume 25, p.4309-4314, (2019) Website
Nitrate and nitrite utilization in sulfate-reducing bacteria, Moura, I., Bursakov S., Costa C., and Moura J. J. , Anaerobe, Oct, Volume 3, Number 5, p.279-90, (1997) AbstractWebsite
Nitric oxide detection using electrochemical third-generation biosensors – based on heme proteins and porphyrins, Gomes, F., Maia L., Cordas C., Delerue-Matos C., Moura I., Moura J. J. G., and Morais S. , Electroanalysis, Volume 30, p.1-20, (2018) Website
Nitric oxide reductase: direct electrochemistry and electrocatalytic activity, Cordas, C. M., Pereira A. S., Martins C. E., Timoteo C. G., Moura I., Moura J. J., and Tavares P. , Chembiochem, Dec, Volume 7, Number 12, p.1878-81, (2006) AbstractWebsite
Nitrite biosensing using cytochrome c nitrite reductase: Towards a disposable strip electrode, Correia, C., Rodrigues M., Silveira C. M., Moura J. J. G., Ochoteco E., Jubete E., and Almeida M. G. , Biomedical Engineering Systems and Technologies. Series: Communications in Computer and Information Science, (2011)
Nitrite biosensing using cytochrome c nitrite reductase: Towards a disposable strip electrode, C., Correia, M. Rodrigues, C.M. Silveira, J.J.G. Moura, E. Ochoteco, E. Jubete, and M.G. Almeida , Biomedical Engineering Systems and Technologies, Communications in Computer and Information Science, DOI:10.1007/978-3-642-38256-7_3, p.41-51, (2013)
Nitrite Biosensing via Selective Enzymes-A Long but Promising Route, Almeida, M. G., Serra A., Silveira C. M., and Moura J. J. , Sensors, Volume 10, Number 12, p.11530-55, (2010) AbstractWebsite

The last decades have witnessed a steady increase of the social and political awareness for the need of monitoring and controlling environmental and industrial processes. In the case of nitrite ion, due to its potential toxicity for human health, the European Union has recently implemented a number of rules to restrict its level in drinking waters and food products. Although several analytical protocols have been proposed for nitrite quantification, none of them enable a reliable and quick analysis of complex samples. An alternative approach relies on the construction of biosensing devices using stable enzymes, with both high activity and specificity for nitrite. In this paper we review the current state-of-the-art in the field of electrochemical and optical biosensors using nitrite reducing enzymes as biorecognition elements and discuss the opportunities and challenges in this emerging market.

Nitrite reduction by molybdoenzymes - A new class of nitric oxide-forming nitrite reductases, Maia, L. B., and Moura J. J. G. , J Biol Inorg Chem, Volume 20, p.403-433, (2015)