Nickel Superoxide Dismutase: Insight Into The Metalloenzyme Gained From Functional Metallopeptide Models

Superoxide (O2*-) is one of the toxic reactive oxygen species produced in the mitochondrial respiratory chain during ATP synthesis. All organisms have evolved defense systems capable of destroing O2*- before it can cause cellular damage. The most widely utilized O2*- detoxification pathway involves metalloenzymes called superoxide dismutases. Based on the metal cofactors present in the active site of the enzymes they are classified into Cu/ZnSOD, MnSOD, FeSOD or NiSOD. Nickel superoxide dismutase (NiSOD) is the most recently discovered SOD, and is found in Streptomyces species and cyanobacteria. The active site of NiSOD utilizes a unique coordination environment. In its reduced state, NiII is coordinated by the N-terminal amine from His1, the amide nitrogen from Cys2, and two cis-thiolates from Cys2 and Cys6 affording a square planar coordination geometry about nickel. Upon oxidation to NiIII, the imidazole of His1 becomes the fifth ligand to nickel affording a square pyramidal geometry about nickel. To understand the contributions of this unique coordination environment utilized by NiSOD we have chosen a metallopeptide based mimicking approach. We have synthesized and characterized a series of synthetic metallopeptide maquettes of NiSOD based on the first 12 amino acid residues from the N-terminus end of S. coelicolor NiSOD. These have been used to investigate the influence of amine/amide vs. bis-amide coordination in NiSOD. Results show that [NiII(SODM1-Ac)] is a poorer SOD than [NiII(SODM1)]. This seems to indicate why nature has chosen the mixture of amine and amide nitrogen ligands at the NiSOD active-site: bis-amide ligation (as is found at the NiN2S2 center of acetyl-coenzyme-A synthase) would produce a poor SOD. We have also probed the role of the axial ligand His(1) on the catalytic activity of NiSOD. We find that His(1) tunes the redox potential towards the mid point potential of the superoxide oxidation and reduction process, and hence accelerates catalysis. Based on our inhibition studies of [NiII(SODM2)] by cyanide and the O2*- mimic azide, superoxide dimutation by NiSOD appears to be taking place by an outer-sphere mechanism. We also probed the influence of some of the non-coordinating amino acid residues in NiSOD to delineate their possible role. Although some amino acids residues such as His(1), Cys(2), Pro(5) and Cys(6) in the Ni-binding hook of NiSOD are conserved, some bacterial species possess variation of the non-coordinating amino acids found in the Ni-binding hook of S. coelicolor NiSOD. Also investigated is the influence of H-bonding of e-imidazole nitrogen of His1. The result shows that H-bonding makes the NiIII-dN weaker, and enhances the catalysis