RESUMEN
Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redox-active metal cycles between oxidized and reduced forms that differ by one electron to catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. Nickel-dependent SOD (NiSOD) is a unique biological solution for controlling superoxide levels. This enzyme relies on the use of cysteinate ligands to bring the Ni(III/II) redox couple into the range required for catalysis (â¼300 mV vs. NHE). The use of cysteine thiolates, which are not found in any other SOD, is a curious choice because of their well-known oxidation by peroxide and dioxygen. The NiSOD active site cysteinate ligands are resistant to oxidation, and prior studies of synthetic and computational models point to the backbone N-donors in the active site (the N-terminal amine and the amide N atom of Cys2) as being involved in stabilizing the cysteines to oxidation. To test the role of the backbone N-donors, we have constructed a variant of NiSOD wherein an alanine residue was added to the N-terminus (Ala0-NiSOD), effectively altering the amine ligand to an amide. X-ray absorption, electronic absorption, and magnetic circular dichroism (MCD) spectroscopic analyses of as-isolated Ala0-NiSOD coupled with density functional theory (DFT) geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and time-dependent DFT computations are consistent with a diamagnetic Ni(II) site with two cysteinate, one His1 amide, and one Cys2 amidate ligands. The variant protein is catalytically inactive, has an altered electronic absorption spectrum associated with the nickel site, and is sensitive to oxidation. Mass spectrometric analysis of the protein exposed to air shows the presence of a mixture of oxidation products, the principal ones being a disulfide, a bis-sulfenate, and a bis-sulfinate derived from the active site cysteine ligands. Details of the electronic structure of the Ni(III) site available from the DFT calculations point to subtle changes in the unpaired spin density on the S-donors as being responsible for the altered sensitivity of Ala0-NiSOD to O2.
Asunto(s)
Amidas/metabolismo , Aminas/metabolismo , Níquel/química , Superóxido Dismutasa/metabolismo , Amidas/química , Aminas/química , Escherichia coli/metabolismo , Regulación Enzimológica de la Expresión Génica , Modelos Moleculares , Conformación Proteica , Superóxido Dismutasa/químicaRESUMEN
Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the NH group of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intrasubunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network and compare the results with prior predictions from density functional theory calculations. The X-ray crystal structure of H1A-NiSOD, which lacks the apical ligand entirely, reveals that in the absence of the Glu17-His1 H-bond, the active site is disordered. Characterization of this variant using X-ray absorption spectroscopy (XAS) shows that Ni(II) is bound in the expected N2S2 planar coordination site. Despite these structural perturbations, the H1A-NiSOD variant retains 4% of wild-type (WT) NiSOD activity. Three other mutations were designed to preserve the apical imidazole ligand but perturb the H-bonding network: R47A-NiSOD, which lacks the intramolecular H-bonding interaction; E17R/R47A-NiSOD, which retains the intramolecular H-bond but lacks the intermolecular Glu17-His1 H-bond; and E17A/R47A-NiSOD, which lacks both H-bonding interactions. These variants were characterized by a combination of techniques, including XAS to probe the nickel site structure, kinetic studies employing pulse-radiolytic production of superoxide, and electron paramagnetic resonance to assess the Ni redox activity. The results indicate that in addition to the roles in redox tuning suggested on the basis of previous computational studies, the Glu17-His1 H-bond plays an important structural role in the proper folding of the "Ni-hook" motif that is a critical feature of the active site.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Níquel/química , Níquel/fisiología , Superóxido Dismutasa/química , Superóxido Dismutasa/fisiología , Sitios de Unión/fisiología , Cristalización , Estructura Secundaria de ProteínaRESUMEN
Nickel-dependent superoxide dismutase (NiSOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O(2) (.-)). A distinctive and fascinating feature of NiSOD is the presence of active-site nickel-thiolate interactions involving the Cys2 and Cys6 residues. Mutation of one or both Cys residues to Ser prevents catalysis of O(2) (.-), demonstrating that both residues are necessary to support proper enzymatic activity (Ryan et al., J Biol Inorg Chem, 2010). In this study, we have employed a combined spectroscopic and computational approach to characterize three Cys-to-Ser (Cys --> Ser) mutants (C2S, C6S, and C2S/C6S NiSOD). Similar electronic absorption and magnetic circular dichroism spectra are observed for these mutants, indicating that they possess nearly identical active-site geometric and electronic structures. These spectroscopic data also reveal that the Ni(2+) ion in each mutant adopts a high-spin (S = 1) configuration, characteristic of a five- or six-coordinate ligand environment, as opposed to the low-spin (S = 0) configuration observed for the four-coordinate Ni(2+) center in the native enzyme. An analysis of the electronic absorption and magnetic circular dichroism data within the framework of density functional theory computations performed on a series of five- and six-coordinate C2S/C6S NiSOD models reveals that the active site of each Cys --> Ser mutant possesses an essentially six-coordinate Ni(2+) center with a rather weak axial bonding interaction. Factors contributing to the lack of catalytic activity displayed by the Cys --> Ser NiSOD mutants are explored.
Asunto(s)
Simulación por Computador , Cisteína/metabolismo , Níquel/química , Serina/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Dominio Catalítico , Dicroismo Circular , Cisteína/química , Cisteína/genética , Ligandos , Modelos Biológicos , Modelos Moleculares , Mutación , Níquel/metabolismo , Serina/química , Serina/genética , Superóxido Dismutasa/genéticaRESUMEN
Nickel superoxide dismutase (NiSOD) is unique among the family of superoxide dismutase enzymes in that it coordinates Cys residues (Cys2 and Cys6) to the redox-active metal center and exhibits a hexameric quaternary structure. To assess the role of the Cys residues with respect to the activity of NiSOD, mutations of Cys2 and Cys6 to Ser (C2S-NiSOD, C6S-NiSOD, and C2S/C6S-NiSOD) were carried out. The resulting mutants do not catalyze the disproportionation of superoxide, but retain the hexameric structure found for wild-type NiSOD and bind Ni(II) ions in a 1:1 stoichiometry. X-ray absorption spectroscopic studies of the Cys mutants revealed that the nickel active-site structure for each mutant resembles that of C2S/C6S-NiSOD and demonstrate that mutation of either Cys2 or Cys6 inhibits coordination of the remaining Cys residue. Mutation of one or both Cys residue(s) in NiSOD induces the conversion of the low-spin Ni(II) site in the native enzyme to a high-spin Ni(II) center in the mutants. This result indicates that coordination of both Cys residues is required to generate the native low-spin configurations and maintain catalytic activity. Analysis of the quaternary structure of the Cys mutants by differential scanning calorimetry, mass spectrometry, and size-exclusion chromatography revealed that the Cys ligands, particularly Cys2, are also important for stabilizing the hexameric quaternary structure of the native enzyme.
Asunto(s)
Cisteína/química , Cisteína/metabolismo , Níquel/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Biocatálisis , Dominio Catalítico , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón , Estabilidad de Enzimas , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Streptomyces coelicolor/enzimología , Superóxido Dismutasa/genética , Espectroscopía de Absorción de Rayos XRESUMEN
Inducible defenses against oxidative stress are coordinated by redox-sensitive transcription factors that transduce oxidative damage into differential gene expression. The opportunistic human pathogen Pseudomonas aeruginosa has evolved under physiological and host-derived sources of oxidative stress. Previous work showed that the pqrABC and pqrR genes of P. aeruginosa, all lacking known functions, were induced by treatment of three different isolates of P. aeruginosa with paraquat (PQ), a superoxide-producing agent. Insertional mutation of the pqrABCR genes resulted in hypersensitive phenotypes to a variety of oxidants, although the hypersensitivity to PQ was marginal. Mutation of pqrR and complementation assays showed that PqrR regulated the pqrABC genes in response to PQ. PqrR, a member of the MarR family of transcriptional regulators, contains a C-terminal region with four conserved cysteines, which suggested redox-regulated transcriptional activity. Purified PqrR bound to two discrete sites at the pqrA and pqrR regulatory regions. The in vitro DNA binding activity of PqrR was decreased by exposure to air and reconstituted by treatment with dl-dithiothreitol. Elemental analysis and preliminary electron paramagnetic resonance experiments showed that PqrR contains iron. Interestingly, site-directed mutagenesis of C-terminal cysteines demonstrated that the four conserved cysteine residues are essential for in vivo redox sensing by PqrR.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Hierro/química , Pseudomonas aeruginosa/metabolismo , Transducción de Señal/fisiología , Elementos Silenciadores Transcripcionales/fisiología , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Diamida/farmacología , Datos de Secuencia Molecular , Mutación , Naftoquinonas/farmacología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Paraquat/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Transcripción GenéticaRESUMEN
Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examined herein for recombinant preparations of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9F/Y62F-, and D3A-NiSOD). Structural aspects of the nickel sites are examined by a combination of EPR and X-ray absorption spectroscopies, and by single-crystal X-ray diffraction at approximately 1.9 A resolution in the case of Y9F- and D3A-NiSODs. The functional effects of the mutations are examined by kinetic studies employing pulse radiolytic generation of O2- and by redox titrations. These studies reveal that although the structure of the nickel center in NiSOD is unique, the ligand environment is designed to optimize the redox potential at 290 mV and results in the oxidation of 50% of the nickel centers in the oxidized hexamer. Kinetic investigations show that all of the mutant proteins have considerable activity. In the case of Y9F-NiSOD, the enzyme exhibits saturation behavior that is not observed in wild-type (WT) NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl- or Br- and is located close to but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addition to affecting the interaction between subunits, this mutation repositions Tyr9 and leads to altered chemistry with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies for adjusting the redox potential and supply of protons are employed, NiSOD has evolved a similar strategy for controlling the access of anions to the active site.