Human mitochondrial manganese superoxide dismutase (MnSOD) catalyzes the dismutation of the superoxide radical anion 2O2(-) + 2H+ -> O2 + H2O2. This catalysis requires cycles of both oxidation and reduction at the metal followed by proton transfers from solution to the active site to release product peroxide. Reactive oxygen species are normally and continuously produced by numerous intracellular processes, including the mitochondrial electron transport chain, and MnSOD is extremely important as a main line of defense against oxidative damage associated with the inflammatory response and postischemic reperfusion of organs. The unifying goal of this proposal is to elucidate the role of active site residues in the catalytic mechanism of human MnSOD, and to emphasize ways to enhance the catalytic activity of MnSOD. The motivation to enhance the efficiency of MnSOD is both to understand the catalysis and to introduce clinical possibilities for gene therapy. The applicant plans structure-function studies using site-directed mutagenesis to alter residues near the active site. Stopped-flow spectrophotometry and pulse radiolysis will be used to evaluate changes in catalysis and x-ray crystallography will be used to detect structural changes. The crystal structure for human MnSOD is known. Investigators have already developed an expression system and prepared wild-type human MnSOD and two significant mutants. Activation will be achieved through several strategies some of which have been useful in other enzymes, particularly the Cu, ZnSOD and carbonic anhydrase: i) enhancement of the proton transfer mechanisms by providing intra- and intermolecular shuttle groups, ii) overcoming product (peroxide) inhibition by altering the active site region through mutagenesis, and iii) enhancement of electrostatic guidance of substrate by altering the charge character near the active site.
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