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Vincent Nivière - One of the best experts on this subject based on the ideXlab platform.
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Iron Hydroperoxide Intermediate in Superoxide Reductase: Protonation or Dissociation First? MM Dynamics and QM/MM Metadynamics Study.
Journal of Chemical Theory and Computation, 2017Co-Authors: Rolf David, Vincent Nivière, Hélène Jamet, Yohann Moreau, Anne MiletAbstract:Superoxide Reductase is a mononuclear iron enzyme involved in Superoxide radical detoxification in some bacteria. Its catalytic mechanism is associated with the remarkable formation of a ferric hydroperoxide Fe3+-OOH intermediate, which is specifically protonated on its proximal oxygen to generate the reaction product H2O2. Here, we present a computational study of the protonation mechanism of the Fe3+-OOH intermediate, at different levels of theory. This was performed on the whole system (solvated protein) using well-tempered metadynamics at the QM/MM (B3LYP/AmberFF99SB) level. Enabled by the development of a new set of force field parameters for the active site, a conformational MM study of the Fe3+-OOH species gave insights into its solvation pattern, in addition to generating the two starting conformations for the ab initio metadynamics setup. Two different protonation mechanisms for the Fe3+-OOH intermediate have been found depending on the starting structure. Whereas a possible mechanism involves at first the protonation of the hydroperoxide ligand and then dissociation of H2O2, the most probable one starts with an unexpected dissociation of the HOO- ligand from the iron, followed by its protonation. This favored reactivity was specifically linked to the influence of both the nearby conserved lysine 48 residue and the microsolvatation on the charge distribution of the oxygens of the HOO- ligand. These data highlight the crucial role of the whole environment, solvent, and protein, to describe accurately this second protonation step in Superoxide Reductase. This is clearly not possible with smaller models unable to reproduce correctly the mechanistically determinant charge distribution.
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Iron Hydroperoxide Intermediate in Superoxide Reductase: Protonation or Dissociation First? MM Dynamics and QM/MM Metadynamics Study.
Journal of chemical theory and computation, 2017Co-Authors: Rolf David, Vincent Nivière, Hélène Jamet, Yohann Moreau, Anne MiletAbstract:Superoxide Reductase is a mononuclear iron enzyme involved in Superoxide radical detoxification in some bacteria. Its catalytic mechanism is associated with the remarkable formation of a ferric hydroperoxide Fe3+-OOH intermediate, which is specifically protonated on its proximal oxygen to generate the reaction product H2O2. Here, we present a computational study of the protonation mechanism of the Fe3+-OOH intermediate, at different levels of theory. This was performed on the whole system (solvated protein) using well-tempered metadynamics at the QM/MM (B3LYP/AmberFF99SB) level. Enabled by the development of a new set of force field parameters for the active site, a conformational MM study of the Fe3+-OOH species gave insights into its solvation pattern, in addition to generating the two starting conformations for the ab initio metadynamics setup. Two different protonation mechanisms for the Fe3+-OOH intermediate have been found depending on the starting structure. Whereas a possible mechanism involves at...
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Identification of iron(III) peroxo species in the active site of the Superoxide Reductase SOR from Desulfoarculus baarsii
arXiv: Chemical Physics, 2015Co-Authors: Christelle Mathé, Murielle Lombard, M. Fontecave, Tony A. Mattioli, Olivier Horner, Jean-marc Latour, Vincent NivièreAbstract:The active site of Superoxide Reductase SOR consists of an Fe2+ center in an unusual [His4 Cys1] square-pyramidal geometry. It specifically reduces Superoxide to produce H2O2. Here, we have reacted the SOR from Desulfoarculus baarsii directly with H2O2. We have found that its active site can transiently stabilize an Fe3+-peroxo species that we have spectroscopically characterized by resonance Raman. The mutation of the strictly conserved Glu47 into alanine results in a stabilization of this Fe3+-peroxo species, when compared to the wild-type form. These data support the hypothesis that the reaction of SOR proceeds through such a Fe3+-peroxo intermediate. This also suggests that Glu47 might serve to help H2O2 release during the reaction with Superoxide.
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Fe3+-hydroxide ligation in the Superoxide Reductase from Desulfoarculus baarsii is associated with pH dependent spectral changes
arXiv: Biomolecules, 2014Co-Authors: Christelle Mathé, Vincent Nivière, Tony A. MattioliAbstract:Superoxide Reductase (SOR) catalyzes the reduction of O2*- to H2O2. Its active site consists of a non-heme Fe2+ center in an unusual square-pyramidal [His4 Cys] coordination. Like many SORs, the electronic absorption band corresponding to the oxidized active site of the SOR from Desulfoarculus baarsii exhibits a pH-dependent alkaline transition changing from ca. 644 to 560 nm as the pH increases and with an apparent pKa of 9.0. Variants in which the conserved amino acids glutamate 47 and lysine 48 were replaced by the neutral residues alanine (E47A) and isoleucine (K48I), respectively, exhibited the same alkaline transition but at lower apparent pKa values of 6.7 and 7.6, respectively. Previous work [Nivi{\`e}re, V.; Asso, M.; Weill, C. O.; Lombard, M.; Guigliarelli, B.; Favaudon, V.; Hou{\'e}e-Levin, C. Biochemistry 2004, 43, 808-818] has shown that this alkaline transition is associated with the protonation/deprotonation of an unidentified base, B-, which is neither E47 nor K48. In this work, we show by resonance Raman spectroscopy that at basic pH a high-spin Fe3+-OH species is formed at the active site. The presence of the HO- ligand was directly associated with an absorption band maximum at 560 nm, whereas upon protonation, the band shifts to 644 nm. With respect to our previous work, B- can be identified with this high-spin Fe3+-OH species, which upon protonation results in a water molecule at the active site. Implications for the SOR catalytic cycle are proposed.
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M{\"o}ssbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of Superoxide Reductase from Desulfoarculus Baarsii. Density functional calculations on related models
arXiv: Chemical Physics, 2014Co-Authors: Olivier Horner, Vincent Nivière, M. Fontecave, Christelle Mathé, Tony A. Mattioli, Jean-marie Mouesca, Jean-louis Oddou, Claudine Jeandey, Pascale Maldivi, Pierre BonvilleAbstract:Superoxide Reductase (SOR) is an Fe protein that catalyzes the reduction of Superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Math{\'e} et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a M{\"o}ssbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the M{\"o}ssbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which M{\"o}ssbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The M{\"o}ssbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction (approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of 2 and axial in the case of 3. This difference originates directly from the presence of the thiolate ligand in 2. A correlation between experimental isomer shifts for Fe(3+) mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for 2 and 3 (0.56 and 0.63 mm/s, respectively). A significant increase of isomer shift value is found upon going from a methylthiolate to a nitrogen ligand for the Fe(3+) ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for 3 is likely to be in the 0.61-0.65 mm/s range [Horner et al. (2002) Eur. J. Inorg. Chem., 3278-3283], the isomer shift value for a high-spin eta(2)-O(2) Fe(3+) complex with an axial thiolate group can be estimated to be in the 0.54-0.58 mm/s range. The occurrence of a side-on peroxo intermediate in SOR is discussed in relation to the recent data published for a side-on peroxo-Fe(3+) species in another biological system [Karlsson et al. (2003) Science 299, 1039-1042].
José J. G. Moura - One of the best experts on this subject based on the ideXlab platform.
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Superoxide Reductase: Different Interaction Modes with its Two Redox Partners
Chembiochem : a European journal of chemical biology, 2013Co-Authors: Rui Almeida, Isabel Moura, José J. G. Moura, Paola Turano, Sofia R. PauletaAbstract:Anaerobic organisms have molecular systems to detoxify reactive oxygen species when transiently exposed to oxygen. One of these systems is Superoxide Reductase, which reduces O2 (.-) to H2 O2 without production of molecular oxygen. In order to complete the reduction of Superoxide anion, Superoxide Reductase requires an electron, delivered by its redox partners, which in Desulfovibrio gigas are rubredoxin and/or desulforedoxin. In this work, we characterized the interaction of Desulfovibrio gigas Superoxide Reductase with both electron donors by using steady-state kinetics, 2D NMR titrations, and backbone relaxation measurements. The rubredoxin surface involved in the electron transfer complex with Superoxide Reductase comprises the solvent-exposed hydrophobic residues in the vicinity of its metal center (Cys9, Gly10, Cys42, Gly43, and Ala44), and a Kd of 3 μM at 59 mM ionic strength was estimated by NMR. The ionic strength dependence of Superoxide-mediated rubredoxin oxidation by Superoxide Reductase has a maximum kapp of (37 ± 12) min(-1) at 157 mM. Relative to the electron donor desulforedoxin, its complex with Superoxide Reductase was not detected by chemical shift perturbation, though this protein is able to transfer electrons to Superoxide Reductase with a maximum kapp of (31 ± 7) min(-1) at an ionic strength of 57 mM. Competition experiments using steady-state kinetics and NMR spectroscopy (backbone relaxation measurements and use of a paramagnetic relaxation enhancement probe) with Fe-desulforedoxin in the presence of (15) N-Zn-rubredoxin showed that these two electron donors compete for the same site on the enzyme surface, as shown in the model structure of the complex generated by using restrained molecular docking calculations. These combined strategies indicate that the two small electron donors bind in different manners, with the desulforedoxin complex being a short lived electron transfer complex or more dynamic, with many equivalent kinetically competent orientations.
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Enzymatic activity mastered by altering metal coordination spheres
JBIC Journal of Biological Inorganic Chemistry, 2008Co-Authors: Isabel Moura, Sofia R. Pauleta, José J. G. MouraAbstract:Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite Reductase, cytochrome c peroxidase and cytochrome cd _1 nitrite Reductase), non-heme proteins (Superoxide Reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide Reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion.
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The first crystal structure of class III Superoxide Reductase from Treponema pallidum
JBIC Journal of Biological Inorganic Chemistry, 2006Co-Authors: Teresa Santos-silva, Francoise Auchere, Patricia Raleiras, Isabel Moura, José J. G. Moura, José Trincão, Ana L Carvalho, Cecília Bonifácio, Maria J RomãoAbstract:Superoxide Reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of Superoxide radicals in the cell. The crystal structure of Treponema pallidum ( Tp ) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl_2 and diffracted beyond 1.55 Å resolution. The overall architecture is very similar to that of other known SORs but Tp SOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the β-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)_4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in Tp SOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the Tp SOR structure also suggest that Lys49 might be involved in attraction of Superoxide to the active site.
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Superoxide Reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays.
Acta crystallographica. Section F Structural biology and crystallization communications, 2005Co-Authors: Teresa Santos-silva, Francoise Auchere, Isabel Moura, José J. G. Moura, José Trincão, Ana L Carvalho, Cecília Bonifácio, Maria J RomãoAbstract:Superoxide Reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging Superoxide radicals from the cell. The oxidized form of Treponema pallidum Superoxide Reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P2(1) (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A, beta = 106.9 degrees) and diffracted beyond 1.60 A resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A, beta = 104.9 degrees) and diffracted beyond 1.55 A. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (lambda = 1.542 A) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2(1) data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.
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Superoxide Reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2005Co-Authors: Teresa Santos-silva, Francoise Auchere, Isabel Moura, José J. G. Moura, José Trincão, Ana L Carvalho, Cecília Bonifácio, Maria J RomãoAbstract:Superoxide Reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging Superoxide radicals from the cell. The oxidized form of Treponema pallidum Superoxide Reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P21 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A, β = 106.9°) and diffracted beyond 1.60 A resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A, β = 104.9°) and diffracted beyond 1.55 A. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (λ = 1.542 A) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P21 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.
Isabel Moura - One of the best experts on this subject based on the ideXlab platform.
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Superoxide Reductase: Different Interaction Modes with its Two Redox Partners
Chembiochem : a European journal of chemical biology, 2013Co-Authors: Rui Almeida, Isabel Moura, José J. G. Moura, Paola Turano, Sofia R. PauletaAbstract:Anaerobic organisms have molecular systems to detoxify reactive oxygen species when transiently exposed to oxygen. One of these systems is Superoxide Reductase, which reduces O2 (.-) to H2 O2 without production of molecular oxygen. In order to complete the reduction of Superoxide anion, Superoxide Reductase requires an electron, delivered by its redox partners, which in Desulfovibrio gigas are rubredoxin and/or desulforedoxin. In this work, we characterized the interaction of Desulfovibrio gigas Superoxide Reductase with both electron donors by using steady-state kinetics, 2D NMR titrations, and backbone relaxation measurements. The rubredoxin surface involved in the electron transfer complex with Superoxide Reductase comprises the solvent-exposed hydrophobic residues in the vicinity of its metal center (Cys9, Gly10, Cys42, Gly43, and Ala44), and a Kd of 3 μM at 59 mM ionic strength was estimated by NMR. The ionic strength dependence of Superoxide-mediated rubredoxin oxidation by Superoxide Reductase has a maximum kapp of (37 ± 12) min(-1) at 157 mM. Relative to the electron donor desulforedoxin, its complex with Superoxide Reductase was not detected by chemical shift perturbation, though this protein is able to transfer electrons to Superoxide Reductase with a maximum kapp of (31 ± 7) min(-1) at an ionic strength of 57 mM. Competition experiments using steady-state kinetics and NMR spectroscopy (backbone relaxation measurements and use of a paramagnetic relaxation enhancement probe) with Fe-desulforedoxin in the presence of (15) N-Zn-rubredoxin showed that these two electron donors compete for the same site on the enzyme surface, as shown in the model structure of the complex generated by using restrained molecular docking calculations. These combined strategies indicate that the two small electron donors bind in different manners, with the desulforedoxin complex being a short lived electron transfer complex or more dynamic, with many equivalent kinetically competent orientations.
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Enzymatic activity mastered by altering metal coordination spheres
JBIC Journal of Biological Inorganic Chemistry, 2008Co-Authors: Isabel Moura, Sofia R. Pauleta, José J. G. MouraAbstract:Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite Reductase, cytochrome c peroxidase and cytochrome cd _1 nitrite Reductase), non-heme proteins (Superoxide Reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide Reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion.
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The first crystal structure of class III Superoxide Reductase from Treponema pallidum
JBIC Journal of Biological Inorganic Chemistry, 2006Co-Authors: Teresa Santos-silva, Francoise Auchere, Patricia Raleiras, Isabel Moura, José J. G. Moura, José Trincão, Ana L Carvalho, Cecília Bonifácio, Maria J RomãoAbstract:Superoxide Reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of Superoxide radicals in the cell. The crystal structure of Treponema pallidum ( Tp ) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl_2 and diffracted beyond 1.55 Å resolution. The overall architecture is very similar to that of other known SORs but Tp SOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the β-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)_4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in Tp SOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the Tp SOR structure also suggest that Lys49 might be involved in attraction of Superoxide to the active site.
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Superoxide Reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays.
Acta crystallographica. Section F Structural biology and crystallization communications, 2005Co-Authors: Teresa Santos-silva, Francoise Auchere, Isabel Moura, José J. G. Moura, José Trincão, Ana L Carvalho, Cecília Bonifácio, Maria J RomãoAbstract:Superoxide Reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging Superoxide radicals from the cell. The oxidized form of Treponema pallidum Superoxide Reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P2(1) (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A, beta = 106.9 degrees) and diffracted beyond 1.60 A resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A, beta = 104.9 degrees) and diffracted beyond 1.55 A. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (lambda = 1.542 A) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2(1) data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.
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Superoxide Reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2005Co-Authors: Teresa Santos-silva, Francoise Auchere, Isabel Moura, José J. G. Moura, José Trincão, Ana L Carvalho, Cecília Bonifácio, Maria J RomãoAbstract:Superoxide Reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging Superoxide radicals from the cell. The oxidized form of Treponema pallidum Superoxide Reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P21 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A, β = 106.9°) and diffracted beyond 1.60 A resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A, β = 104.9°) and diffracted beyond 1.55 A. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (λ = 1.542 A) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P21 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.
Donald M. Kurtz - One of the best experts on this subject based on the ideXlab platform.
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Treponema denticola Superoxide Reductase: in vivo role, in vitro reactivities, and a novel [Fe(Cys)(4)] site.
Biochemistry, 2012Co-Authors: Jonathan D. Caranto, Linda L. Gebhardt, Charles E. Macgowan, Ronald J. Limberger, Donald M. KurtzAbstract:In vitro and in vivo results are presented demonstrating that Superoxide Reductase (SOR) from the air-sensitive oral spirochete, Treponema denticola (Td), is a principal enzymatic scavenger of Superoxide in this organism. This SOR contains the characteristic non-heme [Fe(His)4Cys] active sites. No other metal-binding domain has been annotated for Td SOR. However, we found that Td SOR also accommodates a [Fe(Cys)4] site whose spectroscopic and redox properties resemble those in so-called 2Fe-SORs. Spectroscopic comparisons of the wild type and engineered Cys → Ser variants indicate that three of the Cys ligands correspond to those in [Fe(Cys)4] sites of “canonical” 2Fe-SORs, whereas the fourth Cys ligand residue has no counterpart in canonical 2Fe-SORs or in any other known [Fe(Cys)4] protein. Structural modeling is consistent with iron ligation of the “noncanonical” Cys residue across subunit interfaces of the Td SOR homodimer. The Td SOR was isolated with only a small percentage of [Fe(Cys)4] sites. Howe...
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Reaction of Desulfovibrio vulgaris two-iron Superoxide Reductase with Superoxide: insights from stopped-flow spectrophotometry.
Biochemistry, 2007Co-Authors: Victor W. Huang, Joseph P. Emerson, Donald M. KurtzAbstract:Stopped-flow mixing of the Desulfovibrio vulgaris two-iron Superoxide Reductase (2Fe-SOR) containing the ferrous active site with Superoxide generates a dead time intermediate whose absorption spectrum is identical to that of a putative ferric-hydroperoxo intermediate previously observed by pulse radiolysis. The dead time intermediate is shown to be a product of reaction with Superoxide and to be generated at a much higher proportion of active sites than by pulse radiolysis. This intermediate decays smoothly to the resting ferric active site ( approximately 30 s-1 at 2 degrees C and pH 7) with no other detectable intermediates. Deuterium isotope effects demonstrate that solvent proton donation occurs in the rate-determining step of dead time intermediate decay and that neither of the conserved pocket residues, Glu47 or Lys48, functions as a rate-determining proton donor between pH 6 and pH 8. Fluoride, formate, azide, and phosphate accelerate decay of the dead time intermediate and for azide or fluoride lead directly to ferric-azido or -fluoro complexes of the active site, which inhibit Glu47 ligation. A solvent deuterium isotope effect is observed for the azide-accelerated decay, and the decay rate constants are proportional to the concentrations and pKa values of HX (X- = F-, HCO2-, N3-). These data indicate that the protonated forms of the anions function analogously to solvent as general acids in the rate-determining step. The results support the notion that the ferrous SOR site reacts with Superoxide by an inner sphere process, leading directly to the ferric-hydroperoxo intermediate, and demonstrate that the decay of this intermediate is subject to both specific- and general-acid catalysis.
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Comparing the electronic properties of the low-spin cyano-ferric [Fe(N 4)(Cys)] active sites of Superoxide Reductase and P450cam using ENDOR spectroscopy and DFT calculations
Journal of the American Chemical Society, 2006Co-Authors: Tran Chin Yang, Donald M. Kurtz, Francis E. Jenney, Michael D. Clay, Michael K. Johnson, Rebecca L Mcnaughton, Rangan Krishnan, Michael W W Adams, Brian M HoffmanAbstract:Superoxide Reductase (SOR) and P450 enzymes contain similar [Fe(N)4(SCys)] active sites and, although they catalyze very different reactions, are proposed to involve analogous low-spin (hydro)peroxo-Fe(III) intermediates in their respective mechanisms that can be modeled by cyanide binding. The equatorial FeN4 ligation by four histidine ligands in CN-SOR and the heme in CN-P450cam is directly compared by 14N ENDOR, while the axial Fe−CN and Fe−S bonding is probed by 13C ENDOR of the cyanide ligand and 1Hβ ENDOR measurements to determine the spin density delocalization onto the cysteine sulfur. There are small, but notable, differences in the bonding between Fe(III) and its ligands in the two enzymes. The ENDOR measurements are complemented by DFT computations that support the semiempirical equation used to compute spin densities on metal-coordinated cysteinyl and shed light on bonding changes as the Fe−C−N linkage bends. They further indicate that H bonds to the cysteinyl thiolate sulfur ligand reduce the...
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comparing the electronic properties of the low spin cyano ferric fe n 4 cys active sites of Superoxide Reductase and p450cam using endor spectroscopy and dft calculations
Journal of the American Chemical Society, 2006Co-Authors: Tran Chin Yang, Donald M. Kurtz, Francis E. Jenney, Michael D. Clay, Michael K. Johnson, Rebecca L Mcnaughton, Rangan Krishnan, Michael W W Adams, Brian M HoffmanAbstract:Superoxide Reductase (SOR) and P450 enzymes contain similar [Fe(N)4(SCys)] active sites and, although they catalyze very different reactions, are proposed to involve analogous low-spin (hydro)perox...
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Avoiding high-valent iron intermediates: Superoxide Reductase and rubrerythrin.
Journal of inorganic biochemistry, 2006Co-Authors: Donald M. KurtzAbstract:The Fenton or Fenton-type reaction between aqueous ferrous ion and hydrogen peroxide generates a highly oxidizing species, most often formulated as hydroxyl radical or ferryl ([Fe(IV)O](2+)). Intracellular Fenton-type chemistry can be lethal if not controlled. Nature has, therefore, evolved enzymes to scavenge Superoxide and hydrogen peroxide, the reduced dioxygen species that initiate intracellular Fenton-type chemistry. Two such enzymes found predominantly in air-sensitive bacteria and archaea, Superoxide Reductase (SOR) and rubrerythrin (Rbr), functioning as a peroxidase (hydrogen peroxide Reductase), contain non-heme iron. The iron coordination spheres in these enzymes contain five or six protein ligands from His and Glu residues, and, in the case of SOR, a Cys residue. SOR contains a mononuclear active site that is designed to protonate and rapidly expel peroxide generated as a product of the enzymatic reaction. The ferrous SOR reacts adventitiously but relatively slowly (several seconds to a few minutes) with exogenous hydrogen peroxide, presumably in a Fenton-type reaction. The diferrous active site of Rbr reacts more rapidly with hydrogen peroxide but can divert Fenton-type reactions towards the two-electron reduction of hydrogen peroxide to water. Proximal aromatic residues may function as radical sinks for Fenton-generated oxidants. Fenton-initiated damage to these iron active sites may become apparent only under extremely oxidizing intracellular conditions.
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Identification of iron(III) peroxo species in the active site of the Superoxide Reductase SOR from Desulfoarculus baarsii
arXiv: Chemical Physics, 2015Co-Authors: Christelle Mathé, Murielle Lombard, M. Fontecave, Tony A. Mattioli, Olivier Horner, Jean-marc Latour, Vincent NivièreAbstract:The active site of Superoxide Reductase SOR consists of an Fe2+ center in an unusual [His4 Cys1] square-pyramidal geometry. It specifically reduces Superoxide to produce H2O2. Here, we have reacted the SOR from Desulfoarculus baarsii directly with H2O2. We have found that its active site can transiently stabilize an Fe3+-peroxo species that we have spectroscopically characterized by resonance Raman. The mutation of the strictly conserved Glu47 into alanine results in a stabilization of this Fe3+-peroxo species, when compared to the wild-type form. These data support the hypothesis that the reaction of SOR proceeds through such a Fe3+-peroxo intermediate. This also suggests that Glu47 might serve to help H2O2 release during the reaction with Superoxide.
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M{\"o}ssbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of Superoxide Reductase from Desulfoarculus Baarsii. Density functional calculations on related models
arXiv: Chemical Physics, 2014Co-Authors: Olivier Horner, Vincent Nivière, M. Fontecave, Christelle Mathé, Tony A. Mattioli, Jean-marie Mouesca, Jean-louis Oddou, Claudine Jeandey, Pascale Maldivi, Pierre BonvilleAbstract:Superoxide Reductase (SOR) is an Fe protein that catalyzes the reduction of Superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Math{\'e} et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a M{\"o}ssbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the M{\"o}ssbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which M{\"o}ssbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The M{\"o}ssbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction (approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of 2 and axial in the case of 3. This difference originates directly from the presence of the thiolate ligand in 2. A correlation between experimental isomer shifts for Fe(3+) mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for 2 and 3 (0.56 and 0.63 mm/s, respectively). A significant increase of isomer shift value is found upon going from a methylthiolate to a nitrogen ligand for the Fe(3+) ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for 3 is likely to be in the 0.61-0.65 mm/s range [Horner et al. (2002) Eur. J. Inorg. Chem., 3278-3283], the isomer shift value for a high-spin eta(2)-O(2) Fe(3+) complex with an axial thiolate group can be estimated to be in the 0.54-0.58 mm/s range. The occurrence of a side-on peroxo intermediate in SOR is discussed in relation to the recent data published for a side-on peroxo-Fe(3+) species in another biological system [Karlsson et al. (2003) Science 299, 1039-1042].
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Mössbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of Superoxide Reductase from Desulfoarculus Baarsii. Density functional calculations on related models.
Biochemistry, 2004Co-Authors: Olivier Horner, Vincent Nivière, M. Fontecave, Christelle Mathé, Tony A. Mattioli, Jean-marie Mouesca, Jean-louis Oddou, Claudine Jeandey, Pascale Maldivi, Pierre BonvilleAbstract:Superoxide Reductase (SOR) is an Fe protein that catalyzes the reduction of Superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Mathé et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a Mössbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the Mössbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which Mössbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The Mössbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction ( approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of 2 and axial in the case of 3. This difference originates directly from the presence of the thiolate ligand in 2. A correlation between experimental isomer shifts for Fe(3+) mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for 2 and 3 (0.56 and 0.63 mm/s, respectively). A significant increase of isomer shift value is found upon going from a methylthiolate to a nitrogen ligand for the Fe(3+) ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for 3 is likely to be in the 0.61-0.65 mm/s range [Horner et al. (2002) Eur. J. Inorg. Chem., 3278-3283], the isomer shift value for a high-spin eta(2)-O(2) Fe(3+) complex with an axial thiolate group can be estimated to be in the 0.54-0.58 mm/s range. The occurrence of a side-on peroxo intermediate in SOR is discussed in relation to the recent data published for a side-on peroxo-Fe(3+) species in another biological system [Karlsson et al. (2003) Science 299, 1039-1042].
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Mössbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of Superoxide Reductase from Desulfoarculus Baarsii. Density functional calculations on related models.
Biochemistry, 2004Co-Authors: Olivier Horner, Vincent Nivière, M. Fontecave, Christelle Mathé, Tony A. Mattioli, Jean-marie Mouesca, Jean-louis Oddou, Claudine Jeandey, Pascale Maldivi, Pierre BonvilleAbstract:Superoxide Reductase (SOR) is an Fe protein that catalyzes the reduction of Superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Mathe et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a Mossbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the Mossbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which Mossbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The Mossbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction ( approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of 2 and axial in the case of 3. This difference originates directly from the presence of the thiolate ligand in 2. A correlation between experimental isomer shifts for Fe(3+) mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for 2 and 3 (0.56 and 0.63 mm/s, respectively). A significant increase of isomer shift value is found upon going from a methylthiolate to a nitrogen ligand for the Fe(3+) ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for 3 is likely to be in the 0.61-0.65 mm/s range [Horner et al. (2002) Eur. J. Inorg. Chem., 3278-3283], the isomer shift value for a high-spin eta(2)-O(2) Fe(3+) complex with an axial thiolate group can be estimated to be in the 0.54-0.58 mm/s range. The occurrence of a side-on peroxo intermediate in SOR is discussed in relation to the recent data published for a side-on peroxo-Fe(3+) species in another biological system [Karlsson et al. (2003) Science 299, 1039-1042].
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Discovery of Superoxide Reductase: an historical perspective
JBIC Journal of Biological Inorganic Chemistry, 2004Co-Authors: Vincent Nivière, M. FontecaveAbstract:For more than 30 years, the only enzymatic system known to catalyze the elimination of Superoxide was Superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that Superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against Superoxide has emerged. It was discovered that elimination of Superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named Superoxide Reductase, SOR. Having played a major role in this discovery, we describe here how the concept of Superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.