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2000
Jovanovic, T, Ascenso C, Hazlett KR, Sikkink R, Krebs C, Litwiller R, Benson LM, Moura I, Moura JJ, Radolf JD, Huynh BH, Naylor S, Rusnak F.  2000.  Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase, Sep 15. J Biol Chem. 275:28439-48., Number 37 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.

Moniz, A, Casaca S, Bairrada M, c}ão Moreno C{\c.  2000.  I&D, inova{\c c}ão e fomento de emprego: ideias chave de uma rela{\c c}ão[R&D, innovation and job creation: key-ideas of a relation], Sep. , Number 9667: University Library of Munich, Germany Abstract

The increasing degree of inter-dependency and integration of economy at a global scale motivated the globalization of scientific and technological knowledge from manufacturing, to the marketing and the strategic management of companies that act as protagonists of these processes. Globalization of markets is inter-connected with intensive incorporation of knowledge in economy. In this sense the continuous evolution of the manufacturing industry structure for an increased high intensity technology elements, namely information and communication technologies, implies that these industrial sub-sectors are conditioning the global performance of economy, the productivity gains and as a consequence the levels of economical and employment growth. This study on “R&D, Innovation and Employment Creation” for the Portuguese Observatory of Employment and Vocational Training (IEFP-MQE) is updating information on the articulation between Job creation, innovation, technology, and R&D. A diagnosis of the state of national S&T research and of processes of technological and organizational innovation was made, as well of the interfacing structures between the knowledge sector and the economical activities. Were made 12 case studies at companies with some innovation activities, interviews to unionists and to Professional associations, and public offices. Was made also a survey to a selected sample of technological infrastructures.

Moniz, A, Casaca S, Bairrada M, Moreno C.  2000.  {I&D, inovação e fomento de emprego: ideias chave de uma relação[R&D, innovation and job creation: key-ideas of a relation]}, Sep. , Number 9667: University Library of Munich, Germany Abstract

The increasing degree of inter-dependency and integration of economy at a global scale motivated the globalization of scientific and technological knowledge from manufacturing, to the marketing and the strategic management of companies that act as protagonists of these processes. Globalization of markets is inter-connected with intensive incorporation of knowledge in economy. In this sense the continuous evolution of the manufacturing industry structure for an increased high intensity technology elements, namely information and communication technologies, implies that these industrial sub-sectors are conditioning the global performance of economy, the productivity gains and as a consequence the levels of economical and employment growth. This study on “R&D, Innovation and Employment Creation” for the Portuguese Observatory of Employment and Vocational Training (IEFP-MQE) is updating information on the articulation between Job creation, innovation, technology, and R&D. A diagnosis of the state of national S&T research and of processes of technological and organizational innovation was made, as well of the interfacing structures between the knowledge sector and the economical activities. Were made 12 case studies at companies with some innovation activities, interviews to unionists and to Professional associations, and public offices. Was made also a survey to a selected sample of technological infrastructures.

Caldeira, J, Belle V, Asso M, Guigliarelli B, Moura I, Moura JJ, Bertrand P.  2000.  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, Mar 14. Biochemistry. 39:2700-7., Number 10 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.

Morelli, X, Dolla A, Czjzek M, Palma PN, Blasco F, Krippahl L, Moura JJ, Guerlesquin F.  2000.  Heteronuclear NMR and soft docking: an experimental approach for a structural model of the cytochrome c553-ferredoxin complex, Mar 14. Biochemistry. 39:2530-7., Number 10 AbstractWebsite

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

Brown, K, Tegoni M, Prudencio M, Pereira AS, Besson S, Moura JJ, Moura I, Cambillau C.  2000.  A novel type of catalytic copper cluster in nitrous oxide reductase, Mar. Nat Struct Biol. 7:191-5., Number 3 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.

Morelli, X, Czjzek M, Hatchikian CE, Bornet O, Fontecilla-Camps JC, Palma NP, Moura JJ, Guerlesquin F.  2000.  Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations, Jul 28. J Biol Chem. 275:23204-10., Number 30 AbstractWebsite

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).

Duarte, RO, Archer M, Dias JM, Bursakov S, Huber R, Moura I, Romao MJ, Moura JJ.  2000.  Biochemical/spectroscopic characterization and preliminary X-ray analysis of a new aldehyde oxidoreductase isolated from Desulfovibrio desulfuricans ATCC 27774, Feb 24. Biochem Biophys Res Commun. 268:745-9., Number 3 AbstractWebsite

Aldehyde oxidoreductase (AOR) activity has been found in different sulfate reducing organisms (Moura, J. J. G., and Barata, B. A. S. (1994) in Methods in Enzymology (Peck, H. D., Jr., and LeGall, J., Eds.), Vol. 243, Chap. 4. Academic Press; Romao, M. J., Knablein, J., Huber, R., and Moura, J. J. G. (1997) Prog. Biophys. Mol. Biol. 68, 121-144). The enzyme was purified to homogeneity from extracts of Desulfovibrio desulfuricans (Dd) ATCC 27774, a sulfate reducer that can use sulfate or nitrate as terminal respiratory substrates. The protein (AORDd) is described as a homodimer (monomer, circa 100 kDa), contains a Mo-MCD pterin, 2 x [2Fe-2S] clusters, and lacks a flavin group. Visible and EPR spectroscopies indicate a close similarity with the AOR purified from Desulfovibrio gigas (Dg) (Barata, B. A. S., LeGall, J., and Moura, J. J. G. (1993) Biochemistry 32, 11559-11568). Activity and substrate specificity for different aldehydes were determined. EPR studies were performed in native and reduced states of the enzyme and after treatment with ethylene glycol and dithiothreitol. The AORDd was crystallized using ammonium sulfate as precipitant and the crystals belong to the space group P6(1)22, with unit cell dimensions a = b = 156.4 and c = 177.1 A. These crystals diffract to beyond 2.5 A resolution and a full data set was measured on a rotating anode generator. The data were used to solve the structure by Patterson Search methods, using the model of AORDg.

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

Battistuzzi, G, D'Onofrio M, Borsari M, Sola M, Macedo AL, Moura JJ, Rodrigues P.  2000.  Redox thermodynamics of low-potential iron-sulfur proteins, Dec. J Biol Inorg Chem. 5:748-60., Number 6 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.

George, GN, Pickering IJ, Yu EY, Prince RC, Bursakov SA, Gavel OY, Moura I, Moura JJG.  2000.  A novel protein-bound copper - Molybdenum cluster, Aug 30. Journal of the American Chemical Society. 122:8321-8322., Number 34 AbstractWebsite
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Prudencio, M, Pereira AS, Tavares P, Besson S, Cabrito I, Brown K, Samyn B, Devreese B, Van Beeumen J, Rusnak F, Fauque G, Moura JJ, Tegoni M, Cambillau C, Moura I.  2000.  Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617, Apr 11. Biochemistry. 39:3899-907., Number 14 AbstractWebsite

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

Andrade, SL, Brondino CD, Feio MJ, Moura I, Moura JJ.  2000.  Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491 EPR assignment of the proximal [2Fe-2S] cluster to the Mo site, Apr. Eur J Biochem. 267:2054-61., Number 7 AbstractWebsite

A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate-reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97-kDa homodimer, with 0.6 +/- 0.1 Mo, 3.6 +/- 0.1 Fe and 0.9 +/- 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyses the oxidation of aldehydes to their carboxylic acid form, following simple Michaelis-Menten kinetics, with the following parameters (for benzaldehyde): K(app/m)= 6.65 microM; V app = 13.12 microM.min(-1); k(app/cat) = 0.96 s(-1). Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe-2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of -275 and -325 mV for the Fe/S I and Fe/S II, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic centers, was used to assign the EPR-distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes.

Brown, K, Tegoni M, Prudencio M, Pereira AS, Besson S, Moura JJ, Moura I, Cambillau C.  2000.  A novel type of catalytic copper cluster in nitrous oxide reductase, Apr. Nature Structural Biology. {7}:{191-195}., Number {3}, 75 VARICK ST, 9TH FLR, NEW YORK, NY 10013-1917 USA: NATURE PUBLISHING GROUP Abstract

Nitrous oxide (N(2)O) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N(2)O elimination from the biosphere, N(2)O reductases catalyze the two-electron reduction of N(2)O to N(2). 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 N(2)O reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 Angstrom. 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. N(2)O 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.

Bencini, A, Bianchi A, Lodeiro C, Masotti A, Parola AJ, Pina F, de Melo JS, Valtancoli B.  2000.  A novel fluorescent chemosensor exhibiting exciplex emission. An example of an elementary molecular machine driven by pH and by light, 2000. Chemical Communications. :1639-1640. AbstractWebsite

Coordination/detachment of a pendent functionality in the Zn(ii) complex with a macrocyclic ligand L gives rise to on/off switching of exciplex emission, defining an elementary molecular machine whose movements are driven by both pH and light.

Pina, F, Melo MJ, Maestri M, Passaniti P, Balzani V.  2000.  Artificial chemical systems capable of mimicking some elementary properties of neurons. Journal of the American Chemical Society. 122:4496-4498., Number 18 AbstractWebsite
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Duarte, RO, Archer M, Dias JM, Bursakov S, Huber R, Moura I, Romao MJ, Moura JJG.  2000.  Biochemical/spectroscopic characterization and preliminary X-ray analysis of a new aldehyde oxidoreductase isolated from Desulfovibrio desulfuricans ATCC 27774. Biochemical and Biophysical Research Communications. 268:745-749., Number 3 AbstractWebsite
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Pina, F, Bernardo MA, Garcia-Espana E.  2000.  Fluorescent chemosensors containing polyamine receptors. European Journal of Inorganic Chemistry. :2143-2157., Number 10 AbstractWebsite
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Laia, CAT, Brown W, Almgren M, Costa SMB.  2000.  Light scattering study of water-in-oil AOT microemulsions with poly(oxy)ethylene. Langmuir. 16:465-470., Number 2 AbstractWebsite
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Bazzicalupi, C, Bencini A, Bianchi A, Giorgi C, Fusi V, Masotti A, Valtancoli B, Roque A, Pina F.  2000.  pH modulation of the luminescence emission of a new europium cryptate complex. Chemical Communications. :561-562., Number 7 AbstractWebsite
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Elisei, F, Lima JC, Ortica F, Aloisi GG, Costa M, Leitao E, Abreu I, Dias A, Bonifacio V, Medeiros J, Macanita AL, Becker RS.  2000.  Photophysical properties of hydroxy-substituted flavothiones. Journal of Physical Chemistry a. 104:6095-6102., Number 25 Abstract
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Prudencio, M, Pereira AS, Tavares P, Besson S, Cabrito I, Brown K, Samyn B, Devreese B, Van Beeumen J, Rusnak F, Fauque G, Moura JJG, Tegoni M, Cambillau C, Moura I.  2000.  Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617. Biochemistry. {39}:{3899-3907}., Number {14} Abstract

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named CUA and Cut. Cut could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)= 2.015, A(x) = 1.5 mT, g(y) = 2.071, A(y) = 2 mT, g(z) = 2.138, A(z) = 7 mT) and a strong absorption at similar to 640 nm. Cu-A can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x) g(y) = 2.021, A(x) = A(y) = 0 T, g(z) =0.178, A(z) = 4 mT) and absorption bands at 480, 540, and similar to 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the CuA center. In form A, CUA is predominantly oxidized (S = 1/2, Cu1.5+-Cu1.5+), while in form B it is mostly in the one-electron reduced state (S = 0, Cu1+-Cu1+). In both forms, Cu-Z remains reduced (S = 1/2). Complete crystallographic data at 2.4 Angstrom indicate that Cu-A is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu-Z is a novel tetracopper cluster [Brown, K., et ai. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

1999
Alves, T, Besson S, Duarte LC, Pettigrew GW, Girio FMF, Devreese B, Vandenberghe I, Van Beeumen J, Fauque G, Moura I.  1999.  A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization, Oct 12. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology. 1434:248-259., Number 2 AbstractWebsite

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 mu M and allowing a maximal catalytic centre activity of 116 000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature. (C) 1999 Elsevier Science B.V. All rights reserved.

Marques, MMB, Lobo AM, Prabhakar S, Branco PS.  1999.  A Diels-Alder, retro-Diels-Alder approach to arcyriaflavin-A, MAY 7. TETRAHEDRON LETTERS. 40:3795-3796., Number 19 Abstract
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