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B
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.

C
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)
Carbon dioxide utilisation - bioelectrochemical approaches, C.M., Cordas, J.J.G. Moura, A. Escapa, and R. Mateos , Enzymes for Solving Humankind's Problems, Moura J.J.G., Moura I., Maia L.B. (eds), p.83-108, (2021)
EPR and Mossbauer spectroscopic studies on enoate reductase, Caldeira, J., Feicht R., White H., Teixeira M., Moura J. J., Simon H., and Moura I. , J Biol Chem, Aug 2, Volume 271, Number 31, p.18743-8, (1996) AbstractWebsite

Enoate reductase (EC 1.3.1.31) is a protein isolated from Clostridium tyrobutyricum that contains iron, labile sulfide, FAD, and FMN. The enzyme reduces the alpha,beta carbon-carbon double bond of nonactivated 2-enoates and in a reversible way that of 2-enals at the expense of NADH or reduced methyl viologen. UV-visible and EPR potentiometric titrations detect a semiquinone species in redox intermediate states characterized by an isotropic EPR signal at g = 2.0 without contribution at 580 nm. EPR redox titration shows two widely spread mid-point redox potentials (-190 and -350 mV at pH 7. 0), and a nearly stoichiometric amount of this species is detected. The data suggest the semiquinone radical has an anionic nature. In the reduced form, the [Fe-S] moiety is characterized by a single rhombic EPR spectrum, observed in a wide range of temperatures (4. 2-60 K) with g values at 2.013, 1.943, and 1.860 (-180 mV at pH 7.0). The gmax value is low when compared with what has been reported for other iron-sulfur clusters. Mossbauer studies reveal the presence of a [4Fe-4S]+2/+1 center. One of the subcomponents of the spectrum shows an unusually large value of quadrupole splitting (ferrous character) in both the oxidized and reduced states. Substrate binding to the reduced enzyme induces subtle changes in the spectroscopic Mossbauer parameters. The Mossbauer data together with known kinetic information suggest the involvement of this iron-sulfur center in the enzyme mechanism.

Analysis of the electron paramagnetic resonance properties of the [2Fe-2S]1+ centers in molybdenum enzymes of the xanthine oxidase family: assignment of signals I and II, Caldeira, J., Belle V., Asso M., Guigliarelli B., Moura I., Moura J. J., and Bertrand P. , Biochemistry, Mar 14, Volume 39, Number 10, p.2700-7, (2000) AbstractWebsite

Molybdoenzymes of the xanthine oxidase family contain two [2Fe-2S](1+,2+) clusters that are bound to the protein by very different cysteine motifs. In the X-ray crystal structure of Desulfovibrio gigas aldehyde oxidoreductase, the cluster ligated by a ferredoxin-type motif is close to the protein surface, whereas that ligated by an unusual cysteine motif is in contact with the molybdopterin [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 270, 1170-1176]. These two clusters display distinct electron paramagnetic resonance (EPR) signals: the less anisotropic one, called signal I, is generally similar to the g(av) approximately 1.96-type signals given by ferredoxins, whereas signal II often exhibits anomalous properties such as very large g values, broad lines, and very fast relaxation properties. A detailed comparison of the temperature dependence of the spin-lattice relaxation time and of the intensity of these signals in D. gigas aldehyde oxidoreductase and in milk xanthine oxidase strongly suggests that the peculiar EPR properties of signal II arise from the presence of low-lying excited levels reflecting significant double exchange interactions. The issue raised by the assignment of signals I and II to the two [2Fe-2S](1+) clusters was solved by using the EPR signal of the Mo(V) center as a probe. The temperature dependence of this signal could be quantitatively reproduced by assuming that the Mo(V) center is coupled to the cluster giving signal I in xanthine oxidase as well as in D. gigas aldehyde oxidoreductase. This demonstrates unambiguously that, in both enzymes, signal I arises from the center which is closest to the molybdenum cofactor.

Primary sequence, oxidation-reduction potentials and tertiary-structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin, Caldeira, J., Palma P. N., Regalla M., Lampreia J., Calvete J., Schafer W., Legall J., Moura I., and Moura J. J. , Eur J Biochem, Mar 15, Volume 220, Number 3, p.987-95, (1994) AbstractWebsite

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate-reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, -387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J.J.G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K.D., Sieker, L.C., Jensen, L.H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.

Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas, Cammack, R., Rao K. K., Hall D. O., Moura J. J., Xavier A. V., Bruschi M., Legall J., Deville A., and Gayda J. P. , Biochim Biophys Acta, Feb 22, Volume 490, Number 2, p.311-21, (1977) AbstractWebsite

Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.

ESR studies of cytochrome c3 from Desulfovibrio desulfuricans strain Norway 4: Midpoint potentials of the four haems, and interactions with ferredoxin and colloidal sulphur, Cammack, R., Fauque G., Moura J. J. G., and Legall J. , Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Volume 784, Number 1, p.68-74, (1984) AbstractWebsite
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17O ENDOR detection of a solvent-derived Ni-(OH(x))-Fe bridge that is lost upon activation of the hydrogenase from Desulfovibrio gigas, Carepo, M., Tierney D. L., Brondino C. D., Yang T. C., Pamplona A., Telser J., Moura I., Moura J. J., and Hoffman B. M. , J Am Chem Soc, Jan 16, Volume 124, Number 2, p.281-6, (2002) AbstractWebsite

Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --> Ni-C --> Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.

Hydrogen metabolism in Desulfovibrio desulfuricans strain New Jersey (NCIMB 8313)--comparative study with D. vulgaris and D. gigas species, Carepo, M., Baptista J. F., Pamplona A., Fauque G., Moura J. J., and Reis M. A. , Anaerobe, Dec, Volume 8, Number 6, p.325-32, (2002) AbstractWebsite

This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.

Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis, Carepo, M. S., Carreira C., Grazina R., Zakrzewska M. E., Dolla A., Aubert C., Pauleta S. R., Moura J. J. G., and Moura I. , J Biol Inorg Chem, Volume 21, p.53-62, (2016)
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)
The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase, Carreira, C., Nunes R. F., Mestre O., Moura I., and Pauleta S. R. , J Biol Inorg Chem, Volume 25, p.927, (2020)
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.

The catalytic cycle of nitrous oxide reductase - The enzyme that catalyzes the last step of denitrification, Carreira, C., Pauleta S. R., and Moura I. , J Inorg Biochem, Volume 177, p.423-434, (2017)
Can ultrasonic energy efficiently speed (18)O-labeling of proteins?, Carreira, Ricardo J., Lodeiro Carlos, Diniz Mario S., Moura Isabel, and Capelo Jose L. , Proteomics, Nov, Volume 9, Number 21, p.4974-4977, (2009) AbstractWebsite

We report in this work on the robustness of ultrasonic energy as a tool to speed the isotopic labeling of proteins using the (18)O-decoupling procedure. The first part of the decoupling procedure, comprising protein denaturation, reduction, alkylation and digestion, is done in 8 min under the effects of an ultrasonic field whilst the second part, the isotopic labeling, was assayed with and without the use of ultrasonic energy. Our results clearly demonstrate that the (18)O-isotopic labeling in a decoupling procedure cannot be accelerated using an ultrasonic field.

Can ultrasonic energy efficiently speed (18)O-labeling of proteins?, Carreira, Ricardo J., Lodeiro Carlos, Diniz Mario S., Moura Isabel, and Capelo Jose L. , Proteomics, Nov, Volume 9, Number 21, p.4974-4977, (2009) AbstractWebsite

We report in this work on the robustness of ultrasonic energy as a tool to speed the isotopic labeling of proteins using the (18)O-decoupling procedure. The first part of the decoupling procedure, comprising protein denaturation, reduction, alkylation and digestion, is done in 8 min under the effects of an ultrasonic field whilst the second part, the isotopic labeling, was assayed with and without the use of ultrasonic energy. Our results clearly demonstrate that the (18)O-isotopic labeling in a decoupling procedure cannot be accelerated using an ultrasonic field.

Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes, Carreira, C., Mestre O., Nunes R. F., Moura I., and Pauleta S. R. , PEERJ, Volume 6, p.DOI: 10.7717/peerj.5603, (2018)
Electrode Kinetics of Ion Jelly and Ion Sol-Gel Redox Materials on Screen-Printed Electrodes, Carvalho, R. N. H., Cordas C. M., and Fonseca L. P. , Appl Sci, Volume 12, p.2087, (2022)
Sandwich-Type Enzymatic Fuel Cell Based on a New Electro-Conductive Material - Ion Jelly, Carvalho, R., Almeida R., Moura J. J. G., Lourenço N., Fonseca L., and Cordas C. M. , Chemistry Select, Volume 1, p.6546–6552, (2016) Website
Small phospho-donors phosphorylate MorR without inducing protein conformational changes, Castro, N. S. S., Laia C. A. T., Maiti B. K., Cerqueira N., Moura I., and Carepo M. S. P. , Biophys Chem, Volume 240, p.25-33, (2018)
Fluorescence anisotropy of fluorescein varies according to pH: lessons for binding studies, Castro, N. S. S., Laia C. A. T., Moura I., and Carepo M. S. , J Photochem Photobiol A: Chemistry, Volume 372, p.59-62, (2019)