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Structural aspects of denitrifying enzymes, Moura, I., and Moura J. J. , Curr Opin Chem Biol, Apr, Volume 5, Number 2, p.168-75, (2001) AbstractWebsite

The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

Tungsten-containing formate dehydrogenase from Desulfovibrio gigas: metal identification and preliminary structural data by multi-wavelength crystallography, Raaijmakers, H., Teixeira S., Dias J. M., Almendra M. J., Brondino C. D., Moura I., Moura J. J., and Romao M. J. , J Biol Inorg Chem, Apr, Volume 6, Number 4, p.398-404, (2001) AbstractWebsite

The tungsten-containing formate dehydrogenase (W-FDH) isolated from Desulfovibrio gigas has been crystallized in space group P2(1), with cell parameters a = 73.8 A, b = 111.3 A, c = 156.6 A and beta = 93.7 degrees. These crystals diffract to beyond 2.0 A on a synchrotron radiation source. W-FDH is a heterodimer (92 kDa and 29 kDa subunits) and two W-FDH molecules are present in the asymmetric unit. Although a molecular replacement solution was found using the periplasmic nitrate reductase as a search model, additional phasing information was needed. A multiple-wavelength anomalous dispersion (MAD) dataset was collected at the W- and Fe-edges, at four different wavelengths. Anomalous and dispersive difference data allowed us to unambiguously identify the metal atoms bound to W-FDH as one W atom with a Se-cysteine ligand as well as one [4Fe-4S] cluster in the 92 kDa subunit, and three additional [4Fe-4S] centers in the smaller 29 kDa subunit. The D. gigas W-FDH was previously characterized based on metal analysis and spectroscopic data. One W atom was predicted to be bound to two molybdopterin guanine dinucleotide (MGD) pterin cofactors and two [4Fe-4S] centers were proposed to be present. The crystallographic data now reported reveal a selenium atom (as a Se-cysteine) coordinating to the W site, as well as two extra [4Fe-4S] clusters not anticipated before. The EPR data were re-evaluated in the light of these new results.

Dissimilatory Nitrate Reductase, Romão, M. J., Dias J. M., and Moura I. , Handbook of Metalloproteins , p.1075-1085, (2001) Abstract
Electrochemical studies of rubredoxin from Desulfovibrio vulgaris at modified electrodes, Correia dos Santos, M. M., Paes de Sousa P. M., Simões Gonçalves M. L., Ascenso C., Moura I., and Moura J. J. G. , Journal of Electroanalytical Chemistry, Volume 501, Number 1–2, p.173-179, (2001) AbstractWebsite
Proteómica: a Interface entre a Biologia Molecular e a Biochemistry de Proteínas, Almeida, G., Rodrigues C., and Lampreia J. , Bol. Soc. Port. Química, Volume 82, p.49-56, (2001) Abstract
Redox potential measurements of the Mycobacterium tuberculosis heme protein KatG and the isoniazid-resistant enzyme KatG(S315T): insights into isoniazid activation, Wengenack, N. L., Lopes H., Kennedy M. J., Tavares P., Pereira A. S., Moura I., Moura J. J., and Rusnak F. , Biochemistry, Sep 19, Volume 39, Number 37, p.11508-13, (2000) AbstractWebsite

Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe(3+)/Fe(2+) couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and II or oxyferrous KatG.

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

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

Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774, Rebelo, J., Macieira S., Dias J. M., Huber R., Ascenso C. S., Rusnak F., Moura J. J., Moura I., and Romao M. J. , J Mol Biol, Mar 17, Volume 297, Number 1, p.135-46, (2000) AbstractWebsite

The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate specificity similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6(1)22, with unit cell dimensions of a=b=156.4 A and c=177.1 A, and diffraction data were obtained to beyond 2.8 A. The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is reflected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identifiable as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules. As deduced from this work, both these enzymes are very closely related in terms of their sequences as well as 3D structures. The comparison allowed confirmation and establishment of features that are essential for their function; namely, conserved residues in the active-site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.

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.

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

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.

BiGGER: a new (soft) docking algorithm for predicting protein interactions, Palma, P. N., Krippahl L., Wampler J. E., and Moura J. J. , Proteins, Jun 1, Volume 39, Number 4, p.372-84, (2000) AbstractWebsite

A new computationally efficient and automated "soft docking" algorithm is described to assist the prediction of the mode of binding between two proteins, using the three-dimensional structures of the unbound molecules. The method is implemented in a software package called BiGGER (Bimolecular Complex Generation with Global Evaluation and Ranking) and works in two sequential steps: first, the complete 6-dimensional binding spaces of both molecules is systematically searched. A population of candidate protein-protein docked geometries is thus generated and selected on the basis of the geometric complementarity and amino acid pairwise affinities between the two molecular surfaces. Most of the conformational changes observed during protein association are treated in an implicit way and test results are equally satisfactory, regardless of starting from the bound or the unbound forms of known structures of the interacting proteins. In contrast to other methods, the entire molecular surfaces are searched during the simulation, using absolutely no additional information regarding the binding sites. In a second step, an interaction scoring function is used to rank the putative docked structures. The function incorporates interaction terms that are thought to be relevant to the stabilization of protein complexes. These include: geometric complementarity of the surfaces, explicit electrostatic interactions, desolvation energy, and pairwise propensities of the amino acid side chains to contact across the molecular interface. The relative functional contribution of each of these interaction terms to the global scoring function has been empirically adjusted through a neural network optimizer using a learning set of 25 protein-protein complexes of known crystallographic structures. In 22 out of 25 protein-protein complexes tested, near-native docked geometries were found with C(alpha) RMS deviations < or =4.0 A from the experimental structures, of which 14 were found within the 20 top ranking solutions. The program works on widely available personal computers and takes 2 to 8 hours of CPU time to run any of the docking tests herein presented. Finally, the value and limitations of the method for the study of macromolecular interactions, not yet revealed by experimental techniques, are discussed.

Using cytochrome c(3) to make selenium nanowires, Abdelouas, A., Gong W. L., Lutze W., Shelnutt J. A., Franco R., and Moura I. , Chemistry of Materials, Jun, Volume 12, Number 6, p.1510-+, (2000) AbstractWebsite

We report on a new method to make nanostructures in aqueous solution at room temperature. We used the protein cytochrome c(3) to catalyze reduction of selenate (SeO42-) to selenium Se-0 by dithionite. Reduction was instantaneous. After a week spherical nanoparticles of red Se-0 (about 50 nm diameter) precipitated, followed by self-assembling into crystalline nanowires, typically 1 mu m long. The nanowires were composed of one strand of spherical particles; thicker strands contained several nanoparticles in parallel.

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations, Morelli, X., Czjzek M., Hatchikian C. E., Bornet O., Fontecilla-Camps J. C., Palma N. P., Moura J. J., and Guerlesquin F. , J Biol Chem, Jul 28, Volume 275, Number 30, p.23204-10, (2000) 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).

Biochemical/spectroscopic characterization and preliminary X-ray analysis of a new aldehyde oxidoreductase isolated from Desulfovibrio desulfuricans ATCC 27774, Duarte, R. O., Archer M., Dias J. M., Bursakov S., Huber R., Moura I., Romao M. J., and Moura J. J. , Biochem Biophys Res Commun, Feb 24, Volume 268, Number 3, p.745-9, (2000) 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.

Crystallization and preliminary X-ray analysis of a membrane-bound nitrite reductase from Desulfovibrio desulfuricans ATCC 27774, Dias, J. M., Cunha C. A., Teixeira S., Almeida G., Costa C., Lampreia J., Moura J. J., Moura I., and Romao M. J. , Acta Crystallogr D Biol Crystallogr, Feb, Volume 56, Number Pt 2, p.215-7, (2000) AbstractWebsite

Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A. A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF. However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future.

Evidence for antisymmetric exchange in cuboidal 3Fe-4S (+) clusters, Sanakis, Y., Macedo A. L., Moura I., Moura J. J. G., Papaefthymiou V., and Munck E. , Journal of the American Chemical Society, Dec 6, Volume 122, Number 48, p.11855-11863, (2000) AbstractWebsite

Iron-sulfur clusters with [3Fe-4S] cores are widely distributed in biological systems. In the oxidized state, designated [3Fe-4S](+), these electron-transfer agents have an electronic ground state with S = 1/2, and; they exhibit EPR signals centered at g = 2.01. It has been established by Mossbauer spectroscopy that the three iron sites of the cluster are high-spin Fe3+; and the general properties of the S = 1/2 ground state have been described with the exchange Hamiltonian H-exch = J(12)S(1).S-2 + J(23)S(2).S-3 + J(13)S(1).S-3 Some [3Fe-4S](+) clusters (type 1) have their g-values confined to the range between g = 2.03 and 2.00 while others (type 2) exhibit a continuous distribution of g-values down to g approximate to 1.85. Despite considerable efforts in various laboratories no model has emerged that explains the g-values of type 2 clusters. The 4.2 K spectra of all [3Fe-4S](+) clusters have broad features,which have been simulated in the past by using Fe-57 magnetic hyperfine tensors with anisotropies that are unusually large for high-spin feme sites. It is proposed here that antisymmetric exchange, H-AS = d.(S-1 x S-2 + S-2 x S-3 + S-3 x S-1), is the cause of the g-value shifts in type 2 clusters. We have been able to fit the EPR and Mossbauer spectra of the 3Fe clusters of beef heart aconitase and Desulfovibrio gigas ferredoxin II by using antisymmetric exchange in combination with distributed exchange coupling constants J(12), J(13), and J(23) (J-strain). While antisymmetric exchange is negligible for aconitase (which has a type 1 cluster), fits of the ferredoxin II spectra require \d\ approximate to 0.4 cm(-1). Our studies show that the data of both proteins can lie fit using the same isotropic Fe-57 magnetic hyperfine coupling constant for th three cluster sites, namely a -18.0 MHz for aconitase and a = -18.5 MHz for the D. gigas ferredoxin. The effects of antisymmetric exchange and J-strain on the Mossbauer and EPR spectra are discussed.

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.

Desulfoferrodoxin: a modular protein, Ascenso, C., Rusnak F., Cabrito I., Lima M. J., Naylor S., Moura I., and Moura J. J. , J Biol Inorg Chem, Dec, Volume 5, Number 6, p.720-9, (2000) AbstractWebsite

The gene encoding the non-heme iron-containing desulfoferrodoxin from Desulfovibrio vulgaris was cloned in two fragments in order to obtain polypeptides corresponding to the N- and C-terminal domains observed in the tertiary structure. These fragments were expressed in Escherichia coli, purified to homogeneity and biochemically and spectroscopically characterized. Both recombinant fragments behaved as independent metal-binding domains. The N-terminal fragment exhibited properties similar to desulforedoxin, as expected by the presence of a Fe(S-Cys)4 metal binding motif. The C-terminal fragment, which accommodates a Fe(Nepsilon-His)3(Ndelta-His)(S-Cys) center, was shown to have properties similar to neelaredoxin, except for the reaction with superoxide. The activities of desulfoferrodoxin and of the expressed C-terminal fragment were tested with superoxide in the presence and absence of cytochrome c. The results are consistent with superoxide reductase activity and a possible explanation for the low superoxide consumption in the superoxide dismutase activity assays is proposed.

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

Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491 EPR assignment of the proximal [2Fe-2S] cluster to the Mo site, Andrade, S. L., Brondino C. D., Feio M. J., Moura I., and Moura J. J. , Eur J Biochem, Apr, Volume 267, Number 7, p.2054-61, (2000) 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.

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

Observation of ligand-based redox chemistry at the active site of a molybdenum enzyme, George, G. N., Costa C., Moura J. J. G., and Moura I. , Journal of the American Chemical Society, Mar 24, Volume 121, Number 11, p.2625-2626, (1999) AbstractWebsite