Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent Evolution

Abstract

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-, Y9FY62F- 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 ~ 1.9 Å 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 shows saturation behavior that is not observed in 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 Y9 and leads to altered chemistry with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies are employed to adjust the redox potential and supply of protons, NiSOD has evolved a similar strategy to control the access of anions to the active site.

Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and Its H-Bonding Network

“…The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (W. M. Keck DNA facility at Yale University, New Haven, CT). BL21 (DE3) pLysS (Novagen™) competent cells were transformed with the plasmid containing the desired mutation(s) and the variant protein was expressed and purified as previously described (12, 13) and detailed below.…”

“…(13) Prior to cryo-cooling and data collection, the crystals were transferred into growth buffer supplemented with 40% glycerol. X-ray diffraction data (266 frames, 0.75 degrees per frame) were collected at APS beamline X6A at a wavelength of 0.9184 Å.…”

“…(13, 14, 19) Nickel K -edge XAS data were collected on beamline X3B (and X9B) at the National Synchrotron Light Source (Brookhaven National Laboratory). Samples of frozen protein solutions (1-3 mM, based on nickel content, in 20 mM Tris•HCl, pH 8.0) were placed in polycarbonate holders inserted into aluminum blocks and held near 50 K using a He displex cryostat.…”

“…(13, 14) Superoxide radicals were generated upon pulse radiolysis of an aqueous, air/O 2 saturated solution containing 10 mM phosphate, 30 mM formate, and 5 μM ethylenediaminetetraacetic acid (EDTA). Catalytic rate constants were obtained by monitoring the disappearance of O 2 •− at 260 nm in the presence of micromolar concentrations of mutant NiSOD.…”

Cysteinate Protonation and Water Hydrogen Bonding at the Active-Site of a Nickel Superoxide Dismutase Metallopeptide-Based Mimic: Implications for the Mechanism of Superoxide Reduction

“…2: Ni−N, n = 2, r = 1.913(3) Å, σ = 0.006(1) Å 2 ; ε 2 = 1.08. 14,15 derived bond lengths of NiSOD itself. We note that the addition of a water molecule also reorients the S−H + moiety such that the proton is positioned within the Cys(6)S−Ni−N amine cleft.…”