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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 further investigation of the cytochrome b5-cytochrome c complex, Banci, L., Bertini I., Felli I. C., Krippahl L., Kubicek K., Moura J. J., and Rosato A. , J Biol Inorg Chem, Sep, Volume 8, Number 7, p.777-86, (2003) AbstractWebsite

The interaction of reduced rabbit cytochrome b(5) with reduced yeast iso-1 cytochrome c has been studied through the analysis of (1)H-(15)N HSQC spectra, of (15)N longitudinal ( R(1)) and transverse ( R(2)) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b(5) has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b(5).

Aldehyde oxidoreductase activity in Desulfovibrio gigas: in vitro reconstitution of an electron-transfer chain from aldehydes to the production of molecular hydrogen, Barata, B. A., Legall J., and Moura J. J. , Biochemistry, Nov 2, Volume 32, Number 43, p.11559-68, (1993) AbstractWebsite

The molybdenum [iron-sulfur] protein, first isolated from Desulfovibrio gigas by Moura et al. [Moura, J. J. G., Xavier, A. V., Bruschi, M., Le Gall, J., Hall, D. O., & Cammack, R. (1976) Biochem. Biophys. Res. Commun. 72, 782-789], was later shown to mediate the electronic flow from salicylaldehyde to a suitable electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) [Turner, N., Barata, B., Bray, R. C., Deistung, J., LeGall, J., & Moura, J. J. G. (1987) Biochem. J. 243, 755-761]. The DCPIP-dependent aldehyde oxidoreductase activity was studied in detail using a wide range of aldehydes and analogues. Steady-state kinetic analysis (KM and Vmax) was performed for acetaldehyde, propionaldehyde, benzaldehyde, and salicylaldehyde in excess DCPIP concentration, and a simple Michaelis-Menten model was shown to be applicable as a first kinetic approach. Xanthine, purine, allopurinol, and N1-methylnicotinamide (NMN) could not be utilized as enzyme substrates. DCPIP and ferricyanide were shown to be capable of cycling the electronic flow, whereas other cation and anion dyes [O2 and NAD(P)+] were not active in this process. The enzyme showed an optimal pH activity profile around 7.8. This molybdenum hydroxylase was shown to be part of an electron-transfer chain comprising four different soluble proteins from D. gigas, with a total of 11 discrete redox centers, which is capable of linking the oxidation of aldehydes to the reduction of protons.

Mossbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase, Barata, B. A., Liang J., Moura I., Legall J., Moura J. J., and Huynh B. H. , Eur J Biochem, Mar 1, Volume 204, Number 2, p.773-8, (1992) AbstractWebsite

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. Mossbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. Mossbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.

Redox thermodynamics of low-potential iron-sulfur proteins, Battistuzzi, G., D'Onofrio M., Borsari M., Sola M., Macedo A. L., Moura J. J., and Rodrigues P. , J Biol Inorg Chem, Dec, Volume 5, Number 6, p.748-60, (2000) AbstractWebsite

The enthalpy and entropy changes associated with protein reduction (deltaHdegrees,(rc), deltaSdegrees,(rc)) were determined for a number of low-potential iron-sulfur proteins through variable temperature direct electrochemical experiments. These data add to previous estimates making available, overall, the reduction thermodynamics for twenty species from various sources containing all the different types of metal centers. These parameters are discussed with reference to structural data and calculated electrostatic metal-environment interaction energies, and redox properties of model complexes. This work, which is the first systematic investigation on the reduction thermodynamics of Fe-S proteins, contributes to the comprehension of the determinants of the differences in reduction potential among different protein families within a novel perspective. Moreover, comparison with analogous data obtained previously for electron transport (ET) metalloproteins with positive reduction potentials, i.e., cytochromes c, blue copper proteins, and HiPIPs, helps our understanding of the factors controlling the reduction potential in ET species containing different metal cofactors. The main results of this work can be summarized as follows.

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.

Spectroscopic properties of the cytochrome CD1 from the marine denitrifier Pseudomonas nautica, Besson, S., Carneiro C., Moura J. J. G., Moura I., and Fauque G. , Spectroscopy of Biological Molecules, p.263-264, (1995) AbstractWebsite
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X-ray crystal structure and EPR spectra of "arsenite-inhibited" Desulfovibriogigas aldehyde dehydrogenase: a member of the xanthine oxidase family, Boer, D. R., Thapper A., Brondino C. D., Romao M. J., and Moura J. J. , J Am Chem Soc, Jul 21, Volume 126, Number 28, p.8614-5, (2004) AbstractWebsite

X-ray crystallography has been used to determine the structure of arsenite-inhibited aldehyde dehydrogenase from Desulfovibrio gigas, a member of the xanthine oxidase family of mononuclear molybdenum enzymes. The structure shows an AsO3 moiety bound to the molybdenum atom of the active site through one of the oxygen atoms. A reduced sample of arsenite-inhibited aldehyde dehydrogenase has a Mo(V) signal that shows anisotropic hyperfine and quadrupole coupling to one arsenic atom. This signal has a strong resemblance with a previously reported signal for arsenite-inhibited xanthine oxidase.

Crystallization and preliminary X-ray diffraction analysis of the di-haem cytochrome c peroxidase from Pseudomonas stutzeri, Bonifácio, Cecília, Cunha Carlos A., Müller Axel, Timóteo Cristina G., Dias João M., Moura Isabel, and Romão Maria João , Acta Crystallographica Section D, Volume 59, Number 2, p.345-347, (2003) AbstractWebsite
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Information from e.p.r. spectroscopy on the iron-sulphur centres of the iron-molybdenum protein (aldehyde oxidoreductase) of Desulfovibrio gigas, Bray, R. C., Turner N. A., Legall J., Barata B. A., and Moura J. J. , Biochem J, Dec 15, Volume 280 ( Pt 3), p.817-20, (1991) AbstractWebsite

E.p.r. spectra of reduced iron-sulphur centres of the aldehyde oxidoreductase (iron-molybdenum protein) of Desulfovibrio gigas were recorded at X-band and Q-band frequencies and simulated. Results are consistent with the view that only two types of [2Fe-2S] clusters are present, as in eukaryotic molybdenum-containing hydroxylases. The data indicate the Fe/SI centre to be very similar, and the Fe/SII centre somewhat similar, to these centres in the eukaryotic enzymes.

Effects of bilirubin molecular species on membrane dynamic properties of human erythrocyte membranes: a spin label electron paramagnetic resonance spectroscopy study, Brito, M. A., Brondino C. D., Moura J. J., and Brites D. , Arch Biochem Biophys, Mar 1, Volume 387, Number 1, p.57-65, (2001) AbstractWebsite

Unconjugated bilirubin is a neurotoxic pigment that interacts with membrane lipids. In this study we used electron paramagnetic resonance and the spin labels 5-, 7-, 12-, and 16-doxyl-stearic acid (DSA) to evaluate the depth of the hydrocarbon chain at which interaction of bilirubin preferentially occurs. In addition, we used different pH values to determine the molecular species involved. Resealed right-side-out ghosts were incubated (1-60 min) with bilirubin (3.4-42.8 microM) at pH 7.0, 7.4, and 8.0. Alterations of membrane dynamic properties were maximum after 15 min of incubation with 8.6 microM bilirubin at pH 7.4 and were accompanied by a significant release of phospholipids. Interestingly, concentrations of bilirubin up to 42.8 microM and longer incubations resulted in the elution of cholesterol and further increased that of phospholipids while inducing less structural alterations. Variation of the pH values from 8.0 to 7.4 and 7.0, under conditions of maximum perturbation, led to a change from an increased to a diminished polarity sensed by 5-DSA. Conversely, a progressive enhancement in fluidity was reported by 7-DSA, followed by 12- and 16-DSA. These results indicate that bilirubin while enhancing membrane lipid order at C-5 simultaneously has disordering effects at C-7. Furthermore, recovery of membrane dynamics after 15 min of bilirubin exposure along with the release of lipids is compatible with a membrane adaptive response to the insult. In addition, our data provide evidence that uncharged diacid is the species primarily interacting with the membrane as perturbation is favored by acidosis, a condition frequently associated with hyperbilirubinemia in premature and severely ill infants.

Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria, Brondino, C. D., Passeggi M. C., Caldeira J., Almendra M. J., Feio M. J., Moura J. J., and Moura I. , J Biol Inorg Chem, Mar, Volume 9, Number 2, p.145-51, (2004) AbstractWebsite

We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126+/-2 kDa composed of two subunits, alpha=93+/-3 kDa and beta=32+/-2 kDa, which contains 6+/-1 Fe/molecule, 0.4+/-0.1 Mo/molecule, 0.3+/-0.1 W/molecule, and 1.3+/-0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d(1) configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.

Structural and electron paramagnetic resonance (EPR) studies of mononuclear molybdenum enzymes from sulfate-reducing bacteria, Brondino, C. D., Rivas M. G., Romao M. J., Moura J. J., and Moura I. , Acc Chem Res, Oct, Volume 39, Number 10, p.788-96, (2006) AbstractWebsite

Molybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. The metal atom (Mo or W) is coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. In addition, these proteins contain redox cofactors such as iron-sulfur clusters and heme groups. All of these metal cofactors are along an electron-transfer pathway that mediates the electron exchange between substrate and an external electron acceptor (for oxidative reactions) or donor (for reductive reactions). We describe in this Account a combination of structural and electronic paramagnetic resonance studies that were used to reveal distinct aspects of these enzymes.

Molybdenum and tungsten enzymes: the xanthine oxidase family, Brondino, C. D., Romao M. J., Moura I., and Moura J. J. , Curr Opin Chem Biol, Apr, Volume 10, Number 2, p.109-14, (2006) AbstractWebsite

Mononuclear molybdenum and tungsten are found in the active site of a diverse group of enzymes that, in general, catalyze oxygen atom transfer reactions. Enzymes of the xanthine oxidase family are the best-characterized mononuclear Mo-containing enzymes. Several 3D structures of diverse members of this family are known. Recently, the structures of substrate-bound and arsenite-inhibited forms of two members of this family have also been reported. In addition, spectroscopic studies have been utilized to elucidate fine details that complement the structural information. Altogether, these studies have provided an important amount of information on the characteristics of the active site and the electron transfer pathways.

A novel type of catalytic copper cluster in nitrous oxide reductase, Brown, K., Tegoni M., Prudencio M., Pereira A. S., Besson S., Moura J. J., Moura I., and Cambillau C. , Nat Struct Biol, Mar, Volume 7, Number 3, p.191-5, (2000) AbstractWebsite

Nitrous oxide (N20) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N20 elimination from the biosphere, N20 reductases catalyze the two-electron reduction of N20 to N2. These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N20 reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 A. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N20 binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

Revisiting the catalytic CuZ cluster of nitrous oxide (N2O) reductase. Evidence of a bridging inorganic sulfur, Brown, K., Djinovic-Carugo K., Haltia T., Cabrito I., Saraste M., Moura J. J., Moura I., Tegoni M., and Cambillau C. , J Biol Chem, Dec 29, Volume 275, Number 52, p.41133-6, (2000) AbstractWebsite

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N(2)O to N(2). The crystal structure of N2ORs from Pseudomonas nautica (Pn) and Paracoccus denitrificans (Pd) were solved at resolutions of 2.4 and 1.6 A, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol. 7, 191-195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.

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.

The amino acid sequence of desulforedoxin, a new type of non heme iron protein from Desulfovibrio gigas, Bruschi, M., Moura I., Legall J., Xavier A. V., and Sieker L. C. , Biochemical and Biophysical Research Communications, Volume 90, Number 2, p.596-605, (1979) AbstractWebsite
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Purification, characterization and biological activity of three forms of ferredoxin from the sulfate-reducing bacterium Desulfovibrio gigas, Bruschi, M., Hatchikian C., Legall J., Moura J. J., and Xavier A. V. , Biochim Biophys Acta, Nov 9, Volume 449, Number 2, p.275-84, (1976) AbstractWebsite

Three forms of ferredoxin FdI, FdI', and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI' present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI' and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI' and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.

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.

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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Enzymatic properties and effect of ionic strength on periplasmic nitrate reductase (NAP) from Desulfovibrio desulfuricans ATCC 27774, Bursakov, S. A., Carneiro C., Almendra M. J., Duarte R. O., Caldeira J., Moura I., and Moura J. J. , Biochem Biophys Res Commun, Oct 29, Volume 239, Number 3, p.816-22, (1997) AbstractWebsite

Some sulfate reducing bacteria can induce nitrate reductase when grown on nitrate containing media being involved in dissimilatory reduction of nitrate, an important step of the nitrogen cycle. Previously, it was reported the purification of the first soluble nitrate reductase from a sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (S.A. Bursakov, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, and J.J.G. Moura (1995) Anaerobe 1, 55-60). The present work provides further information about this monomeric periplasmic nitrate reductase (Dd NAP). It has a molecular mass of 74 kDa, 18.6 U specific activity, KM (nitrate) = 32 microM and a pHopt in the range 8-9.5. Dd NAP has peculiar properties relatively to ionic strength and cation/anion activity responses. It is shown that monovalent cations (potassium and sodium) stimulate NAP activity and divalent (magnesium and calcium) inhibited it. Sulfate anion also acts as an activator in KPB buffer. NAP native form is protected by phosphate anion from cyanide inactivation. In the presence of phosphate, cyanide even stimulates NAP activity (up to 15 mM). This effect was used in the purification procedure to differentiate between nitrate and nitrite reductase activities, since the later is effectively blocked by cyanide. Ferricyanide has an inhibitory effect at concentrations higher than 1 mM. The N-terminal amino acid sequence has a cysteine motive C-X2-C-X3-C that is most probably involved in the coordination of the [4Fe-4S] center detected by EPR spectroscopy. The active site of the enzyme consists in a molybdopterin, which is capable for the activation of apo-nit-1 nitrate reductase of Neurospora crassa. The oxidized product of the pterin cofactor obtained by acidic hidrolysis of native NAP with sulfuric acid was identified by HPLC chromatography and characterized as a molybdopterin guanine dinucleotide (MGD).

Isolation and preliminary characterization of a soluble nitrate reductase from the sulfate reducing organism Desulfovibrio desulfuricans ATCC 27774, Bursakov, S., Liu M. Y., Payne W. J., Legall J., Moura I., and Moura J. J. , Anaerobe, Feb, Volume 1, Number 1, p.55-60, (1995) AbstractWebsite

Desulfovibrio desulfuricans ATCC 27774 is a sulfate reducer that can adapt to nitrate respiration, inducing the enzymes required to utilize this alternative metabolic pathway. Nitrite reductase from this organism has been previously isolated and characterized, but no information was available on the enzyme involved in the reduction of nitrate. This is the first report of purification to homogeneity of a nitrate reductase from a sulfate reducing organism, thus completing the enzymatic system required to convert nitrate (through nitrite) to ammonia. D. desulfuricans nitrate reductase is a monomeric (circa 70 kDa) periplasmic enzyme with a specific activity of 5.4 K(m) for nitrate was estimated to be 20 microM. EPR signals due to one [4Fe-4S] cluster and Mo(V) were identified in dithionite reduced samples and in the presence of nitrate.

Antagonists Mo and Cu in a heterometallic cluster present on a novel protein (orange protein) isolated from Desulfovibrio gigas, Bursakov, S. A., Gavel O. Y., Di Rocco G., Lampreia J., Calvete J., Pereira A. S., Moura J. J., and Moura I. , J Inorg Biochem, May, Volume 98, Number 5, p.833-40, (2004) AbstractWebsite

An orange-coloured protein (ORP) isolated from Desulfovibrio gigas, a sulphate reducer, has been previously shown by extended X-ray absorption fine structure (EXAFS) to contain a novel mixed-metal sulphide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)] [J. Am. Chem. Soc. 122 (2000) 8321]. We report here the purification and the biochemical/spectroscopic characterisation of this novel protein. ORP is a soluble monomeric protein (11.8 kDa). The cluster is non-covalently bound to the polypeptide chain. The presence of a MoS(4)(2-) moiety in the structure of the cofactor contributes with a quite characteristic UV-Vis spectra, exhibiting an orange colour, with intense absorption peaks at 480 and 338 nm. Pure ORP reveals an Abs(480)/Abs(338) ratio of 0.535. The gene sequence coding for ORP as well as the amino acid sequence was determined. The putative biological function of ORP is discussed.