Investigation of the Structure/Function Relationship in Nickel Containing Superoxide Dismutase

Reactive oxygen species (ROSs) are a byproduct of aerobic metabolism and inflammation, generated during radiation, and products of metal-catalyzed reactions. ROSs include hydrogen peroxide, superoxide, and hydroxyl radicals and at low cellular concentrations ROSs participate in non-harmful cellular signaling systems. However, if not removed from the cell ROSs can cause significant damage to lipids, membranes, proteins and nucleic acids at high cellular concentrations. The harmful effects of ROSs are balanced by the antioxidant action of non-enzymatic and enzymatic antioxidants. Superoxide dismutases are responsible for detoxifying the cell of superoxide by metabolizing it to oxygen and hydrogen peroxide. To date four types of superoxide dismutases are known; Cu/ZnSOD, FeSOD, MnSOD, and NiSOD. The goal of this research is to investigate how nature has adapted to utilize nickel in order to catalyze the disproportionation reaction of superoxide and the specific roles played by the protein matrix to achieve SOD activity. In the active site of NiSOD, the metal cofactor is bound to the protein by the N-terminal amine, back bone amide of Cys2, imidazole side chain of His1, and the side chain thiolates of Cys2 and Cys6. Sulfur-donor ligands such as thiolates are implicated in activating nickel redox in biochemistry, and as such all known and structurally characterized nickel enzymes can be separated into two classes; those which contain S-donors and redox active nickel centers, and those which contain only N/O-donors and do not participate in nickel centered redox reactions. Although metal-thiolate bonds are highly susceptible to sulfur-based modifications by strong oxidants such as superoxide and hydrogen peroxide; the cysteinate ligands of NiSOD are not oxidized during catalysis. The research reported in this dissertation is focused on mutational analysis of active site nickel ligand residues and second coordination sphere residues that participate in protein interactions with the His1 side chain imidazole within the active site. First coordination sphere mutations C2S, C6S, C2/6S, H1A, Ala0 and second coordination sphere mutation R47A, E17A/R47A, and E17R/R47E have been produced and characterized. It is clear from the mutations in the first coordination sphere that nature has designed the active site of NiSOD that in a way facilitates SOD activity. The Cys2 and Cys6 mutations bind nickel using only N/O-donors and are inactive catalysts in the disproportionation reaction of superoxide. The single C2S and C6S are nearly identical to the double mutant, C2S/C6S, indicating an all-or-none requirement for S-donors in NiSOD. Both S-donors are required to support the native low-spin Ni(II) electronic configuration and the loss of either S-donor leads to conversion to a high-spin Ni(II) center and loss of the remaining S-donor ligand. Mutation of the N-donors, imidazole in H1A and N-terminal amine in Ala0, result in structures which cannot accommodate a stable Ni(III) form of the active site in the as-isolated enzyme and as a result have decreased catalytic activity. Mutations in the second coordination sphere that alter the H-bonding network between His1 and Glu17/Arg47, also result in a destabilized Ni(III)-His on conformation of the active site when the H-bonding network is adjusted (R47A and E17R/R47E) or removed entirely (E17A/R47A)