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MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association, Brown, K., Nurizzo D., Besson S., Shepard W., Moura J., Moura I., Tegoni M., and Cambillau C. , J Mol Biol, Jun 18, Volume 289, Number 4, p.1017-28, (1999) AbstractWebsite

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely. The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit. The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.

Magnetization of the oxidized and reduced three-iron cluster of Desulfovibrio gigas ferredoxin II, Day, E. P., Peterson J., Bonvoisin J. J., Moura I., and Moura J. J. , J Biol Chem, Mar 15, Volume 263, Number 8, p.3684-9, (1988) AbstractWebsite

The saturation magnetizations of the three iron cluster of ferredoxin II of Desulfovibrio gigas in both the oxidized and reduced states have been studied at fixed magnetic fields up to 4.5 tesla over the temperature range from 1.8 to 200 K. The low field (0.3 tesla) susceptibility of oxidized ferredoxin II obeys the Curie law over this entire temperature range. This establishes -2Jox greater than 200 cm-1 as the lower limit for the antiferromagnetic exchange coupling of oxidized ferredoxin II. The saturation magnetizations of reduced ferredoxin II at several fixed fields yield a nested family of curves which can be fit with spin S = 2 and D = -2.7(4) cm-1 (with E/D assigned the value 0.23 as determined by Mossbauer and EPR spectra). The low field susceptibility of reduced ferredoxin II also obeys the Curie law from approximately 4 up to 200 K. This establishes -2Jred greater than 40 cm-1 as the lower limit for the antiferromagnetic coupling of reduced ferredoxin II.

Mammalian ferrochelatase, a new addition to the metalloenzyme family, Ferreira, G. C., Franco R., Lloyd S. G., Pereira A. S., Moura I., Moura J. J., and Huynh B. H. , J Biol Chem, Mar 11, Volume 269, Number 10, p.7062-5, (1994) AbstractWebsite

A [2Fe-2S] cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of [2Fe-2S]+ cluster. Mossbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in 57Fe-enriched medium, demonstrated unambiguously that the cluster is a [2Fe-2S] cluster. No change in the cluster oxidation state was observed during catalysis. The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the [2Fe-2S] center in regulation of mammalian ferrochelatases.

Marinobacter hydrocarbonoclasticus is an aerobic denitrifier, Pauleta, S. R., Ramos S., Pietsch M., Carreira C., Dell'Acqua S., and Moura I. , EuroBIC 11, Granada, p.49-53, (2013)
Measuring the cytochrome c nitrite reductase activity-practical considerations on the enzyme assays, Silveira, C. M., Besson S., Moura I., Moura J. J., and Almeida M. G. , Bioinorg Chem Appl, (2010) AbstractWebsite

The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force.

The mechanism of formate oxidation by metal-dependent formate dehydrogenases, Mota, C. S., Rivas M. G., Brondino C. D., Moura I., Moura J. J., Gonzalez P. J., and Cerqueira N. M. , J Biol Inorg Chem, Dec, Volume 16, Number 8, p.1255-68, (2011) AbstractWebsite

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer, de Sousa, P. M., Pauleta S. R., Goncalves M. L., Pettigrew G. W., Moura I., Dos Santos M. M., and Moura J. J. , J Biol Inorg Chem, Jun, Volume 12, Number 5, p.691-8, (2007) AbstractWebsite

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E (0)', of 230 +/- 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 +/- 0.2 x 10(5) M(-1) s(-1) was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes, Sola, S., Brito M. A., Brites D., Moura J. J. G., and Rodrigues C. M. P. , Clinical Science, Nov, Volume 103, Number 5, p.475-485, (2002) AbstractWebsite

The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.

Metabolic adaptations induced by long-term fasting in quails, Sartori, D. R., Migliorini R. H., Veiga J. A., Moura J. L., Kettelhut I. C., and Linder C. , Comp Biochem Physiol A Physiol, Jul, Volume 111, Number 3, p.487-93, (1995) AbstractWebsite

After up to 21 days without food, adult male quails (Coturnix coturnix japonica) lost about 45% of the initial body weight (100-150 g). As in naturally fast-adapted and larger birds, three phases were identified during prolonged fasting in quails. Phase I lasted 2-3 days and was characterized by a rapid decrease in the rate of body weight loss and high fat mobilization. Phase II was longer and characterized by a slow and steady decline in the rates of body weight loss and of nitrogen excretion. The third (critical) period was marked by an abrupt increase in the rates of body weight loss and of nitrogen excretion. Despite their small size, the duration of phase II in quails was relatively long, a clear advantage for the study of the relationships between the several metabolic events that occur during this crucial adaptative period. Also, the beginning of phase III could be precisely determined. Changes in blood glucose, plasma FFA and triacylglycerols levels, as well as in liver and carcass lipid content were similar to those found in other species of birds. Therefore, quails seem to be a suitable model to investigate the biochemical mechanisms involved in the metabolic adjustments to prolonged food deprivation in non fasting-adapted birds.

Metal binding to the tetrathiolate motif of desulforedoxin and related polypeptides, Kennedy, M., Yu L., Lima M. J., Ascenso C. S., Czaja C., Moura I., Moura J. J. G., and Rusnak F. , Journal of Biological Inorganic Chemistry, Dec, Volume 3, Number 6, p.643-649, (1998) AbstractWebsite

Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)(4) metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe3+ Cd2+, and Zn2+ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe3+ bound with the highest affinity, whereas the affinity of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe3+ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe3+ is reduced to Fe2+ with attendant oxidation of ligand thiols.

Metal ion binding of copper(II), zinc(II) and lead(II) to cytochrome C, Simões Gonçalves, M. L. S., Lopes da Conceição A. C., and Moura J. J. G. , Electrochimica Acta, Volume 35, Number 2, p.473-478, (1990) AbstractWebsite
Metalloenzymes of the denitrification pathway, Tavares, P., Pereira A. S., Moura J. J., and Moura I. , J Inorg Biochem, Dec, Volume 100, Number 12, p.2087-100, (2006) AbstractWebsite

Denitrification, or dissimilative nitrate reduction, is an anaerobic process used by some bacteria for energy generation. This process is important in many aspects, but its environmental implications have been given particular relevance. Nitrate accumulation and release of nitrous oxide in the atmosphere due to excess use of fertilizers in agriculture are examples of two environmental problems where denitrification plays a central role. The reduction of nitrate to nitrogen gas is accomplished by four different types of metalloenzymes in four simple steps: nitrate is reduced to nitrite, then to nitric oxide, followed by the reduction to nitrous oxide and by a final reduction to dinitrogen. In this manuscript we present a concise updated review of the bioinorganic aspects of denitrification.

Metallothioneins and trace elements in digestive gland, gills, kidney and gonads of Octopus vulgaris, Raimundo, J., Costa P. M., Vale C., Costa M. H., and Moura I. , Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, Aug, Volume 152, Number 2, p.139-146, (2010) AbstractWebsite

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (mu g g(-1), dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation. (C) 2010 Elsevier Inc. All rights reserved.

The methylenetetrahydrofolate reductase (MTHFR) 677C-->T mutation and cardiovascular risk--A case of ischemic stroke and acute myocardial infarction, Melo, M., Gaspar E., Madeira S., de Moura P., Alexandrino B., and de Moura J. J. , Rev Port Cardiol, Jan, Volume 24, Number 1, p.89-99, (2005) AbstractWebsite

The authors report the case of a 39-year-old male patient who had an ischemic stroke (complete infarction of right anterior cerebral circulation) and an acute myocardial infarction during the same year. Molecular study revealed he was homozygous for the 677C-->T mutation in the gene coding for methylenetetrahydrofolate reductase, a key enzyme of folate metabolism; deficiency of this enzyme is associated with increased cardiovascular risk and neurological lesions. Some considerations are put forward about hyperhomocysteinemia and the MTHFR 677C-->T mutation as cardiovascular risk factors.

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases, Moura, J. J., Brondino C. D., Trincao J., and Romao M. J. , J Biol Inorg Chem, Oct, Volume 9, Number 7, p.791-9, (2004) AbstractWebsite

Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.

Mo-Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas, Carepo, M. S., Pauleta S. R., Wedd A. G., Moura J. J. G., and Moura I. , J Biol Inorg Chem, Volume 19, p.605-614, (2014)
Modeling protein complexes with BiGGER, Krippahl, L., Moura J. J., and Palma P. N. , Proteins, Jul 1, Volume 52, Number 1, p.19-23, (2003) AbstractWebsite

This article describes the method and results of our participation in the Critical Assessment of PRediction of Interactions (CAPRI) experiment, using the protein docking program BiGGER (Bimolecular complex Generation with Global Evaluation and Ranking) (Palma et al., Proteins 2000;39:372-384). Of five target complexes (CAPRI targets 2, 4, 5, 6, and 7), only one was successfully predicted (target 6), but BiGGER generated reasonable models for targets 4, 5, and 7, which could have been identified if additional biochemical information had been available.

Modelling metallothionein induction in the liver of Sparus aurata exposed to metal-contaminated sediments, Costa, P. M., Repolho T., Caeiro S., Diniz M. E., Moura I., and Costa M. H. , Ecotoxicology and Environmental Safety, Sep, Volume 71, Number 1, p.117-124, (2008) AbstractWebsite

Metallothionein (MT) in the liver of gilthead seabreams (Sparus aurata L., 1758) exposed to Sado estuary (Portugal) sediments was quantified to assess the MT induction potential as a biomarker of sediment-based contamination by copper (Cu), cadmium (U), lead (Pb) and arsenic (As). Sediments were collected from two control sites and four sites with different levels of contamination. Sediment Cu, Cd, Pb, As, total organic matter (TOM) and fine fraction (FF) levels were determined. Generalized linear models (GLM) allowed integration of sediment parameters with liver Cu, Cd, Pb, As and MT concentrations. Although sediment metal levels were lower than expected, we relate NIT with liver Cd and also with interactions between liver and sediment Cu and between liver Cu and TOM. We suggest integrating biomarkers and environmental parameters using statistical models such as GLM as a more sensitive and reliable technique for sediment risk assessment than traditional isolated biomarker approaches. (C) 2007 Elsevier Inc. All rights reserved.

Modelling the electron-transfer complex between aldehyde oxidoreductase and flavodoxin, Krippahl, Ludwig, Palma Nuno P., Moura Isabel, and Moura Jose J. G. , European Journal of Inorganic Chemistry, Oct 2, Number 19, p.3835-3840, (2006) AbstractWebsite

Three-dimensional protein structures of the xanthine oxidase family show different solutions for the problem of transferring electrons between the flavin adenine dinucleotide (FAD) group and the molybdenum cofactor. In xanthine oxidase all the cofactors he within domains of the same protein chain, whereas in CO dehydrogenase the Fe-S centres, FAD and Mo cofactors are enclosed in separate chains and the enzyme exists as a stable complex of all three. In aldehyde oxidore-ductase, only Fe-S and Mo co-factors are present in a single protein chain. Flavodoxin is docked to aldehyde oxidoreductase to mimic the flavin component on the intramolecular electron transfer chain of aanthine oxidase and CO dehydrogenase and, remarkably, the main features of the electron-transfer pathway are observed.

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
Molecular aspects of denitrification/nitrate dissimilation, Moura, I., Cabrito I., Almeida G., Cunha C., Romao M. J., and Moura J. J. G. , Journal of Inorganic Biochemistry, Jul 15, Volume 96, Number 1, p.195-195, (2003) AbstractWebsite
Molecular cloning and sequence analysis of the gene of the molybdenum-containing aldehyde oxido-reductase of Desulfovibrio gigas. The deduced amino acid sequence shows similarity to xanthine dehydrogenase, Thoenes, U., Flores O. L., Neves A., Devreese B., Van Beeumen J. J., Huber R., Romao M. J., Legall J., Moura J. J., and Rodrigues-Pousada C. , Eur J Biochem, Mar 15, Volume 220, Number 3, p.901-10, (1994) AbstractWebsite

In this report, we describe the isolation of a 4020-bp genomic PstI fragment of Desulfovibrio gigas harboring the aldehyde oxido-reductase gene. The aldehyde oxido-reductase gene spans 2718 bp of genomic DNA and codes for a protein with 906 residues. The protein sequence shows an average 52% (+/- 1.5%) similarity to xanthine dehydrogenase from different organisms. The codon usage of the aldehyde oxidoreductase is almost identical to a calculated codon usage of the Desulfovibrio bacteria.

Molybdenum and tungsten enzymes: a brief overview, Cordas, C. M., and Moura J. J. G. , Coord Chem Rev, Volume 394, p.53-64, (2019)
Molybdenum and tungsten enzymes: from biology to chemistry and back, Moura, J. J. G., Bernhardt P. V., Maia L. B., and Gonzalez P. J. , J Biol Inorg Chem, Volume 20, p.181-182, (2015)