Catalytic Mechanism of Human Mn Superoxide Dismutase

（申请人描述）人线粒体超氧化物歧化酶

DESCRIPTION: (Applicant's Description) Human mitochondrial superoxide dismutase (MnSOD) catalyzes the dismutation of the superoxide radical anion, 2O2- + 2H+ - 02 + H202, and is important as a main line of defense against oxidative damage in normal metabolism and in a number of disease states. This catalysis requires proton transfers ultimately from solution to the active site to release product hydrogen peroxide, and is accompanied by a prominent product inhibition. The unifying goals of this proposal are to elucidate the function of active-site residues in the catalytic mechanism and in the product inhibition emphasizing the role of proton transfer and the function of an envelope of hydrophobic residues that surround the metal. We plan structure-function studies using site-specific mutagenesis to alter residues near the active site. Pulse radiolysis and scanning stopped-flow spectrophotometry will be used to evaluate changes in catalysis and x-ray crystallography to direct structural changes. We will also investigate the function of these residues in the fine tuning of the redox potential. Potentiometric titrations will be performed to determine the reduction midpoint potentials of mutants and to discern the influence of active site residues, information which will also be related to the rates of catalysis and extent of product inhibition. Catalysis by MnSOD will be activated by enhancing the rate of protein transfer to the active site using exogenous proton donors and intramolecular shuttle groups. Marcus rate theory will be applied to these data to determine the intrinsic kinetic barrier and thermodynamic components of proton transfer in MnSOD, data that will be compared with proton transfers in other systems.