Role of conserved tyrosine residues in NiSOD catalysis: a case of convergent evolution.

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.

Spectroscopic and computational studies of Ni superoxide dismutase: electronic structure contributions to enzymatic function.

Ni-containing superoxide dismutase (NiSOD) is the most recently discovered member of the class of metalloenzymes that detoxify the superoxide radical in aerobic organisms. In this study, we have employed a variety of spectroscopic and computational methods to probe the electronic structure of the NiSOD active site in both its oxidized (NiSOD(ox), possessing a low-spin (S = (1)/(2)) Ni(3+) center) and reduced (NiSOD(red), containing a diamagnetic Ni(2+) center) states. Our experimentally validated computed electronic-structure description for NiSOD(ox) reveals strong sigma-bonding interactions between Ni and the equatorial S/N ligands, which give rise to intense charge-transfer transitions in the near-UV region of the absorption spectrum. Resonance Raman (rR) spectra obtained with laser excitation in this region exhibit two features at 349 and 365 cm(-)(1) that are assigned to Ni-S(Cys) stretching modes. The NiSOD(red) active site also exhibits a high degree of metal-ligand bond covalency as well as filled/filled pi-interactions between Ni and S/N orbitals, which serve to adjust the redox potential of the Ni(2+) center. Comparison of our computational results for NiSOD(red) with those obtained in parallel studies of synthetic [NiS(2)N(2)] complexes reveals that the presence of an anionic N-donor ligand is crucial for promoting metal-based (versus S-based) oxidation of the active site. The implications of our electronic-structure descriptions with respect to the function of NiSOD are discussed, and a comparison of M-S(Cys) bonding in NiSOD and other metalloenzymes with sulfur ligation is provided.

S K-edge X-ray absorption spectroscopic investigation of the Ni-containing superoxide dismutase active site: new structural insight into the mechanism.

Superoxide dismutases protect cells from the toxic effects of reactive oxygen species derived from superoxide. Nickel-containing superoxide dismutases (NiSOD), found in Streptomyces species and in cyanobacteria, are distinct from Mn-, Fe-, or Cu/Zn-containing SODs in amino acid sequence and metal ligand environment. Sulfur K-edge X-ray absorption spectroscopic investigations were carried out for a series of mono- and binuclear Ni model compounds with varying sulfur ligation, and for oxidized and reduced NiSOD to elucidate the types of Ni-S interactions found in the two oxidation states. The S K-edge XAS spectra clearly indicate the presence of Ni(III)-bound terminal thiolate in the oxidized enzyme and the absence of such coordination to Ni(II) in the peroxide-reduced enzyme. This striking change in the S ligation for Ni with redox suggests that, upon peroxide reduction, an electron is transferred to the Ni(III) site and the terminal thiolate becomes protonated, providing an efficient mechanism for proton-coupled electron transfer.

Expression, reconstitution, and mutation of recombinant Streptomycescoelicolor NiSOD.

Nickel-dependent superoxide dismutases (NiSODs) represent a novel solution to controlling the deleterious effects of reactive oxygen species derived from superoxide in biology. The expression of recombinant Streptomyces coelicolor NiSOD and its in vitro processing and reconstitution to yield fully active enzyme is reported. The results of studies of NiSODs involving mutations in two putative nickel binding ligands are also reported. These studies show that mutation of M28, a strictly conserved residue and one of only three S-donor ligands in the enzyme, has no measurable effect on the spectroscopic or catalytic properties of the enzyme. In contrast, mutation of the strictly conserved N-terminal H residue has dramatic effects on both the spectroscopic and catalytic properties. These results provide insights into structural and mechanistic aspects of the novel nickel-containing reactive site.

Inner-sphere complexation of cobalt(II) 2,9-dimethyl-1,10-phenanthroline ([Co(neo)]2+) with commercial and sol-gel derived silica gel surfaces.

[Co(2,9-dimethyl-1,10-phenanthroline)(solvent)4]2+ ([Co(neo)]2+) undergoes a significant decrease in symmetry to form an inner-sphere surface complex when grafted directly on performed silica or introduced during the sol-gel process. The visible and X-ray absorption spectra of the surface adducts are interpreted in terms of a binding mode in which the Co(II) center has a highly distorted pseudo-C2v symmetry. The interaction of [Co(neo)]2+ with the silica surface was analyzed using an acid-base equilibrium relationship. Half-maximal surface binding was observed at pH ca. 6. Linear fits to the pH dependence data are consistent with inner-sphere binding of a single silanol group to the cobalt center. The formation of the surface species in tetramethoxyorthosilicate (TMOS) sol-gels required approximately 2 equiv of hydroxide anion per cobalt center, suggesting a two-proton-dependent binding event to form a species such as [Co(neo)(SiO)2]. Both sol-gel and silica samples showed essentially identical visible and X-ray absorption spectra, indicating formation of very similar surface adducts when the different synthesis procedures were employed. The maximal binding of [Co(neo)]2+ on three silica samples with different pore diameters and surface areas was compared. Increased binding was found to be inversely proportional to surface area and proportional to pore diameter, indicating a preference for less sterically demanding surface sites.

Pulsed ENDOR and ESEEM Study of [Bis(maleonitriledithiolato)nickel](-): An Investigation into the Ligand Electronic Structure.

By a combination of Q-band pulsed ENDOR (electron nuclear double resonance) and X-band ESEEM (electron stimulated echo envelope modulation) techniques, we have determined the hyperfine tensors for ethylene (C1) and cyano (C2) carbons and N, of [Ni(mnt)(2)](-), along with the quadrupole tensor for nitrogen. These measurements give pi electron spin densities of rho(C1) approximately 0.03 in the C1 2p(z)() orbital, rho(C2) < 0.003, rho(N) approximately 0.01, such that in total, approximately 0.15 of the spin resides on the ligand atoms C and N, while the rest resides in the NiS(4) core, giving rho(NiS(4)(-)) = 0.85. These results are compared with extended Hückel and density functional (BLYP) MO calculations, as well as with Xalpha calculations reported earlier.