The importance of understanding interactomes makes preeminent the study of protein interactions a... more The importance of understanding interactomes makes preeminent the study of protein interactions and protein complexes. Traditionally, protein interactions have been elucidated by experimental methods or, with lower impact, by simulation with protein docking algorithms. This article describes features and applications of the BiGGER docking algorithm, which stands at the interface of these two approaches. BiGGER is a user-friendly docking algorithm that was specifically designed to incorporate experimental data at different stages of the simulation, to either guide the search for correct structures or help evaluate the results, in order to combine the reliability of hard data with the convenience of simulations. Herein, the applications of BiGGER are described by illustrative applications divided in three Case Studies: (Case Study A) in which no specific contact data is available; (Case Study B) when different experimental data (e.g., site-directed mutagenesis, properties of the complex, NMR chemical shift perturbation mapping, electron tunneling) on one of the partners is available; and (Case Study C) when experimental data are available for both interacting surfaces, which are used during the search and/or evaluation stage of the docking. This algorithm has been extensively used, evidencing its usefulness in a wide range of different biological research fields.
Metalloenzymes are the most proficient nature catalysts that are responsible for diverse biochemi... more Metalloenzymes are the most proficient nature catalysts that are responsible for diverse biochemical transformations introducing excellent selectivity and performing at high rates, using intricate mutual relationships between metal ions and proteins. Inspired by nature, chemists started using naturally occurring proteins as templates to harbor non-native metal catalysts for the sustainable synthesis of molecules for pharmaceutical, biotechnological and industrial purposes. Therefore, metalloenzymes are the relevant targets for the design of artificial biocatalysts. The search and development of new scaffolds capable of hosting metals with high levels of selectivity could significantly expand the scope of bio-catalysis. To meet this challenge, herein, three native scaffolds: [1Fe-4Cys] (rubredoxin), [3Fe-4S] (ferredoxin), and [S2MoS2CuS2MoS2]-ORP (orange protein) protein scaffolds are case studies describing templates for the synthesis of non-native monomeric to mixed metal–sulfur cl...
Química 132-Jan-mar 14 artigoS Os metais de transição, essenciais à vida, formam complexos muito ... more Química 132-Jan-mar 14 artigoS Os metais de transição, essenciais à vida, formam complexos muito estáveis com as cadeias laterais dos resíduos de aminoácidos que compõem as proteínas, definindo centros metálicos com geometrias definidas. Complexos com esferas de coordenação totalmente preenchidas não são propensos à interacção com substratos, pelo que, na maioria destes casos, é antecipada a função de transferência electrónica (centros de transferência electrónica). Contudo, em alguns casos, estes complexos constituem centros catalíticos inactivos. Complexos com esferas de coordenação incompletas, ou contendo grupos lábeis, podem formar centros catalíticos activos, que poderão coordenar substratos e activar reacções conducentes à formação de produtos. Desta forma, a actividade enzimática pode ser controlada por alterações da esfera de coordenação do centro metálico catalítico. A conversão de formas inactivas em formas activas pode ser despoletada pela alteração do número de coordenação, acompanhada por alterações conformacionais, criando posições de coordenação disponíveis para os substratos. Serão discutidos exemplos que demonstram claramente que a esfera de coordenação do metal controla a actividade enzimática e, mais ainda, que mostram a dinâmica da transformação das espécies inactivas com consequentes rearranjos estruturais. Um tema comum será delineado onde ligandos e electrões controlam a actividade de metaloproteínas usando conceitos da Química de Coordenação.
In this article, quantum mechanical/molecular mechanical (QM/MM) methods were used to study the f... more In this article, quantum mechanical/molecular mechanical (QM/MM) methods were used to study the full catalytic mechanism of xanthine oxidase (XO).
Bioelectrochemistry (Amsterdam, Netherlands), Jan 21, 2018
Understanding the direct electron transfer processes between redox proteins and electrode surface... more Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b and non-heme Fe, respectively. The determined sensitivity (-4.00 × 10 ± 1.84 × 10 A/μM and - 2.71 × 10 ± 1.44 × 10 A/μM for NO and O, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O, respecti...
In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion produ... more In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochr...
The Orange Protein (ORP) is a small bacterial protein, of unknown function, that harbors a unique... more The Orange Protein (ORP) is a small bacterial protein, of unknown function, that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S2Mo(VI)S2Cu(I)S2Mo(VI)S2](3-), noncovalently bound. The apo-ORP is able to promote the formation and stabilization of this cluster, using Cu(II)- and Mo(VI)S4(2-) salts as starting metallic reagents, to yield a Mo/Cu-ORP that is virtually identical to the native ORP. In this work, we explored the ORP capability of promoting protein-assisted synthesis to prepare novel protein derivatives harboring molybdenum heterometallic clusters containing iron, cobalt, nickel, or cadmium in place of the "central" copper (Mo/Fe-ORP, Mo/Co-ORP, Mo/Ni-ORP, or Mo/Cd-ORP). For that, the previously described protein-assisted synthesis protocol was extended to other metals and the Mo/M-ORP derivatives (M = Cu, Fe, Co, Ni, or Cd) were spectroscopically (UV-visible and electron paramagnetic resonance (EPR)) characterized. The Mo/Cu-ORP and Mo/Cd-OR...
Cytochrome cd1 nitrite reductases (cd 1 NiRs) catalyze the one-electron reduction of nitrite to n... more Cytochrome cd1 nitrite reductases (cd 1 NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd 1 NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd 1 NiR from Marinobacter hydrocarbonoclasticus (Mhcd 1) depends on the presence of its physiological redox partner, cytochrome c 552 (cyt c 552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd 1 and cyt c 552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd 1 and cyt c552 are coimmobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag
The complex [Ph 4 P] 2 [Cu(bdt) 2 ] (1 red) was synthesized by the reaction of [Ph 4 P] 2 [S 2 Mo... more The complex [Ph 4 P] 2 [Cu(bdt) 2 ] (1 red) was synthesized by the reaction of [Ph 4 P] 2 [S 2 MoS 2 CuCl] with H 2 bdt (bdt = benzene-1,2-dithiolate) in basic medium. 1 red is highly susceptible toward dioxygen, affording the one electron oxidized diamagnetic compound [Ph 4 P][Cu(bdt) 2 ] (1 ox). The interconversion between these two oxidation states can be switched by addition of O 2 or base (Et 4 NOH = tetraethylammonium hydroxide), as demonstrated by cyclic voltammetry and UV−visible and EPR spectroscopies. Thiomolybdates, in free or complex forms with copper ions, play an important role in the stability of 1 red during its synthesis, since in its absence, 1 ox is isolated. Both 1 red and 1 ox were structurally characterized by X-ray crystallography. EPR experiments showed that 1 red is a Cu(II)−sulfur complex and revealed strong covalency on the copper−sulfur bonds. DFT calculations confirmed the spin density delocalization over the four sulfur atoms (76%) and copper (24%) atom, suggesting that 1 red has a "thiyl radical character". Time dependent DFT calculations identified such ligand to ligand charge transfer transitions. Accordingly, 1 red is better described by the two isoelectronic structures [Cu I (bdt 2 , 4S 3−, *)] 2− ↔ [Cu II (bdt 2 , 4S 4−)] 2−. On thermodynamic grounds, oxidation of 1 red (doublet state) leads to 1 ox singlet state, [Cu III (bdt 2 , 4S 4−)] 1− .
The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor an... more The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDSPAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at !"C, pH 7.2, using isopropanol and MgC1, as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6,22 or its enantiomorph, with cell dimensions u = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 X nm'/Da (2.5 A3/Da), consistent with the experimental crystal density, p = 1.14 g/cm3. One dimer (approximately 2 X 100 kDa) is located on a crystallographic twofold axis.
JBIC Journal of Biological Inorganic Chemistry, 2006
In this work we present a kinetic study of the superoxide-mediated electron transfer reactions be... more In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) and D. gigas (Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum (Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k app values, 176.9±25.0 min À1 in the case of the Tp/Tp electron transfer, 31.8±3.6 min À1 for the Dg/ Dg electron transfer, and 6.9±1.3 min À1 for Dv/Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron-sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k app value of 108.8±12.0 min À1 , and was then assigned as the potential physiological electron donor in this organism.
JBIC Journal of Biological Inorganic Chemistry, 2011
This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing... more This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.
JBIC Journal of Biological Inorganic Chemistry, 2006
Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed o... more Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe 3 S 4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII int) with four 1 H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII ox and FdII int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1 H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe-4S] core in the oxidized state (DgFdII ox) was used here for the assignment of the same three ligands in the intermediate state (DgFdII int). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm À1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII int. This study, together with the spin delocalization mechanism proposed here for DgFdII int , allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII ox and DgFdII int .
JBIC Journal of Biological Inorganic Chemistry, 2011
Identifying redox partners and the interaction surfaces is crucial for fully understanding electr... more Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N 2 OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N 2 OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N 2 OR, or an electrostatic nature, in the case of P. denitrificans N 2 OR and A. cycloclastes N 2 OR. A set of well-conserved residues on the N 2 OR surface were identified as being part of the electron transfer pathway from the redox partner to N 2 OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N 2 OR sequence). Moreover, we built a model for Wolinella succinogenes N 2 OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N 2 OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N 2 OR domain is similar to that found in the other electron transfer complexes.
JBIC Journal of Biological Inorganic Chemistry, 2004
Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reductio... more Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O : 2 into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, e=6.9±0.4 mM À1 cm À1. Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was À76±5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na 2 IrCl 6oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k app =180 min À1. Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k app =4.5 min À1. Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.
A novel iron Á/sulfur containing protein, a ferredoxin (Fd), was purified to homogeneity from the... more A novel iron Á/sulfur containing protein, a ferredoxin (Fd), was purified to homogeneity from the extract of Desulfovibrio desulfuricans American type culture collection (ATCC) 27774. The purified protein is a 13.4 kDa homodimer with a polypeptide chain of 60 amino acids residues, containing eight cysteines that coordinate two [4Fe Á/4S] clusters. The protein is shown to be air sensitive and cluster conversions take place. We structurally characterize a redox state that contains two [4Fe Á/4S] cores. 1D and 2D 1 H NMR studies are reported on form containing the clusters in the oxidized state. Based on the nuclear Overhauser effect (NOE), relaxation measurements and comparison of the present data with the available spectra of the analogous 8Fe Fds, the cluster ligands were specifically assigned to the eight-cysteinyl residues.
The importance of understanding interactomes makes preeminent the study of protein interactions a... more The importance of understanding interactomes makes preeminent the study of protein interactions and protein complexes. Traditionally, protein interactions have been elucidated by experimental methods or, with lower impact, by simulation with protein docking algorithms. This article describes features and applications of the BiGGER docking algorithm, which stands at the interface of these two approaches. BiGGER is a user-friendly docking algorithm that was specifically designed to incorporate experimental data at different stages of the simulation, to either guide the search for correct structures or help evaluate the results, in order to combine the reliability of hard data with the convenience of simulations. Herein, the applications of BiGGER are described by illustrative applications divided in three Case Studies: (Case Study A) in which no specific contact data is available; (Case Study B) when different experimental data (e.g., site-directed mutagenesis, properties of the complex, NMR chemical shift perturbation mapping, electron tunneling) on one of the partners is available; and (Case Study C) when experimental data are available for both interacting surfaces, which are used during the search and/or evaluation stage of the docking. This algorithm has been extensively used, evidencing its usefulness in a wide range of different biological research fields.
Metalloenzymes are the most proficient nature catalysts that are responsible for diverse biochemi... more Metalloenzymes are the most proficient nature catalysts that are responsible for diverse biochemical transformations introducing excellent selectivity and performing at high rates, using intricate mutual relationships between metal ions and proteins. Inspired by nature, chemists started using naturally occurring proteins as templates to harbor non-native metal catalysts for the sustainable synthesis of molecules for pharmaceutical, biotechnological and industrial purposes. Therefore, metalloenzymes are the relevant targets for the design of artificial biocatalysts. The search and development of new scaffolds capable of hosting metals with high levels of selectivity could significantly expand the scope of bio-catalysis. To meet this challenge, herein, three native scaffolds: [1Fe-4Cys] (rubredoxin), [3Fe-4S] (ferredoxin), and [S2MoS2CuS2MoS2]-ORP (orange protein) protein scaffolds are case studies describing templates for the synthesis of non-native monomeric to mixed metal–sulfur cl...
Química 132-Jan-mar 14 artigoS Os metais de transição, essenciais à vida, formam complexos muito ... more Química 132-Jan-mar 14 artigoS Os metais de transição, essenciais à vida, formam complexos muito estáveis com as cadeias laterais dos resíduos de aminoácidos que compõem as proteínas, definindo centros metálicos com geometrias definidas. Complexos com esferas de coordenação totalmente preenchidas não são propensos à interacção com substratos, pelo que, na maioria destes casos, é antecipada a função de transferência electrónica (centros de transferência electrónica). Contudo, em alguns casos, estes complexos constituem centros catalíticos inactivos. Complexos com esferas de coordenação incompletas, ou contendo grupos lábeis, podem formar centros catalíticos activos, que poderão coordenar substratos e activar reacções conducentes à formação de produtos. Desta forma, a actividade enzimática pode ser controlada por alterações da esfera de coordenação do centro metálico catalítico. A conversão de formas inactivas em formas activas pode ser despoletada pela alteração do número de coordenação, acompanhada por alterações conformacionais, criando posições de coordenação disponíveis para os substratos. Serão discutidos exemplos que demonstram claramente que a esfera de coordenação do metal controla a actividade enzimática e, mais ainda, que mostram a dinâmica da transformação das espécies inactivas com consequentes rearranjos estruturais. Um tema comum será delineado onde ligandos e electrões controlam a actividade de metaloproteínas usando conceitos da Química de Coordenação.
In this article, quantum mechanical/molecular mechanical (QM/MM) methods were used to study the f... more In this article, quantum mechanical/molecular mechanical (QM/MM) methods were used to study the full catalytic mechanism of xanthine oxidase (XO).
Bioelectrochemistry (Amsterdam, Netherlands), Jan 21, 2018
Understanding the direct electron transfer processes between redox proteins and electrode surface... more Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b and non-heme Fe, respectively. The determined sensitivity (-4.00 × 10 ± 1.84 × 10 A/μM and - 2.71 × 10 ± 1.44 × 10 A/μM for NO and O, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O, respecti...
In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion produ... more In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochr...
The Orange Protein (ORP) is a small bacterial protein, of unknown function, that harbors a unique... more The Orange Protein (ORP) is a small bacterial protein, of unknown function, that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S2Mo(VI)S2Cu(I)S2Mo(VI)S2](3-), noncovalently bound. The apo-ORP is able to promote the formation and stabilization of this cluster, using Cu(II)- and Mo(VI)S4(2-) salts as starting metallic reagents, to yield a Mo/Cu-ORP that is virtually identical to the native ORP. In this work, we explored the ORP capability of promoting protein-assisted synthesis to prepare novel protein derivatives harboring molybdenum heterometallic clusters containing iron, cobalt, nickel, or cadmium in place of the "central" copper (Mo/Fe-ORP, Mo/Co-ORP, Mo/Ni-ORP, or Mo/Cd-ORP). For that, the previously described protein-assisted synthesis protocol was extended to other metals and the Mo/M-ORP derivatives (M = Cu, Fe, Co, Ni, or Cd) were spectroscopically (UV-visible and electron paramagnetic resonance (EPR)) characterized. The Mo/Cu-ORP and Mo/Cd-OR...
Cytochrome cd1 nitrite reductases (cd 1 NiRs) catalyze the one-electron reduction of nitrite to n... more Cytochrome cd1 nitrite reductases (cd 1 NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd 1 NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd 1 NiR from Marinobacter hydrocarbonoclasticus (Mhcd 1) depends on the presence of its physiological redox partner, cytochrome c 552 (cyt c 552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd 1 and cyt c 552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd 1 and cyt c552 are coimmobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag
The complex [Ph 4 P] 2 [Cu(bdt) 2 ] (1 red) was synthesized by the reaction of [Ph 4 P] 2 [S 2 Mo... more The complex [Ph 4 P] 2 [Cu(bdt) 2 ] (1 red) was synthesized by the reaction of [Ph 4 P] 2 [S 2 MoS 2 CuCl] with H 2 bdt (bdt = benzene-1,2-dithiolate) in basic medium. 1 red is highly susceptible toward dioxygen, affording the one electron oxidized diamagnetic compound [Ph 4 P][Cu(bdt) 2 ] (1 ox). The interconversion between these two oxidation states can be switched by addition of O 2 or base (Et 4 NOH = tetraethylammonium hydroxide), as demonstrated by cyclic voltammetry and UV−visible and EPR spectroscopies. Thiomolybdates, in free or complex forms with copper ions, play an important role in the stability of 1 red during its synthesis, since in its absence, 1 ox is isolated. Both 1 red and 1 ox were structurally characterized by X-ray crystallography. EPR experiments showed that 1 red is a Cu(II)−sulfur complex and revealed strong covalency on the copper−sulfur bonds. DFT calculations confirmed the spin density delocalization over the four sulfur atoms (76%) and copper (24%) atom, suggesting that 1 red has a "thiyl radical character". Time dependent DFT calculations identified such ligand to ligand charge transfer transitions. Accordingly, 1 red is better described by the two isoelectronic structures [Cu I (bdt 2 , 4S 3−, *)] 2− ↔ [Cu II (bdt 2 , 4S 4−)] 2−. On thermodynamic grounds, oxidation of 1 red (doublet state) leads to 1 ox singlet state, [Cu III (bdt 2 , 4S 4−)] 1− .
The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor an... more The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDSPAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at !"C, pH 7.2, using isopropanol and MgC1, as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6,22 or its enantiomorph, with cell dimensions u = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 X nm'/Da (2.5 A3/Da), consistent with the experimental crystal density, p = 1.14 g/cm3. One dimer (approximately 2 X 100 kDa) is located on a crystallographic twofold axis.
JBIC Journal of Biological Inorganic Chemistry, 2006
In this work we present a kinetic study of the superoxide-mediated electron transfer reactions be... more In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) and D. gigas (Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum (Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k app values, 176.9±25.0 min À1 in the case of the Tp/Tp electron transfer, 31.8±3.6 min À1 for the Dg/ Dg electron transfer, and 6.9±1.3 min À1 for Dv/Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron-sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k app value of 108.8±12.0 min À1 , and was then assigned as the potential physiological electron donor in this organism.
JBIC Journal of Biological Inorganic Chemistry, 2011
This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing... more This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.
JBIC Journal of Biological Inorganic Chemistry, 2006
Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed o... more Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe 3 S 4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII int) with four 1 H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII ox and FdII int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1 H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe-4S] core in the oxidized state (DgFdII ox) was used here for the assignment of the same three ligands in the intermediate state (DgFdII int). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm À1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII int. This study, together with the spin delocalization mechanism proposed here for DgFdII int , allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII ox and DgFdII int .
JBIC Journal of Biological Inorganic Chemistry, 2011
Identifying redox partners and the interaction surfaces is crucial for fully understanding electr... more Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N 2 OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N 2 OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N 2 OR, or an electrostatic nature, in the case of P. denitrificans N 2 OR and A. cycloclastes N 2 OR. A set of well-conserved residues on the N 2 OR surface were identified as being part of the electron transfer pathway from the redox partner to N 2 OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N 2 OR sequence). Moreover, we built a model for Wolinella succinogenes N 2 OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N 2 OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N 2 OR domain is similar to that found in the other electron transfer complexes.
JBIC Journal of Biological Inorganic Chemistry, 2004
Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reductio... more Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O : 2 into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, e=6.9±0.4 mM À1 cm À1. Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was À76±5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na 2 IrCl 6oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k app =180 min À1. Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k app =4.5 min À1. Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.
A novel iron Á/sulfur containing protein, a ferredoxin (Fd), was purified to homogeneity from the... more A novel iron Á/sulfur containing protein, a ferredoxin (Fd), was purified to homogeneity from the extract of Desulfovibrio desulfuricans American type culture collection (ATCC) 27774. The purified protein is a 13.4 kDa homodimer with a polypeptide chain of 60 amino acids residues, containing eight cysteines that coordinate two [4Fe Á/4S] clusters. The protein is shown to be air sensitive and cluster conversions take place. We structurally characterize a redox state that contains two [4Fe Á/4S] cores. 1D and 2D 1 H NMR studies are reported on form containing the clusters in the oxidized state. Based on the nuclear Overhauser effect (NOE), relaxation measurements and comparison of the present data with the available spectra of the analogous 8Fe Fds, the cluster ligands were specifically assigned to the eight-cysteinyl residues.
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Papers by José Moura