Catalytic mechanism and redox properties of human manganese superoxide dismutase.

摘要

Human manganese superoxide dismutase (Mn-SOD) is a detoxifying enzyme in mitochondria that converts superoxide (O2·- ) into oxygen (O2) and hydrogen peroxide (H2O 2). This reaction requires proton and electron transfer between the active site metal and superoxide. Although it is catalytically very active, human Mn-SOD quickly becomes product inhibited by peroxide. The goal of this work is to use site-specific mutagenesis and characterization of the resulting mutants to understand the role in catalysis and inhibition of two prominent active-site residues, Histidine 30 (His30) and Glutamme 143 (Gln143). To do so, I used a variety of techniques including X-ray crystallography, scanning calorimetry, pulse radiolysis, and stopped-low spectrophotometry, performed in our laboratory and through collaborations. In addition, I designed, ordered, and installed the appropriate equipment to measure the redox potential of human Mn-SOD in our laboratory.;Crystallography showed that mutations at both sites 30 and 143 interrupted the hydrogen-bonded network around the metal which possibly affected proton transfer during catalysis. The mutant His30Asn remained uninhibited during catalysis and could eliminate superoxide more efficiently than the wild type enzyme. Mutations at position 143 had a profound effect on both the kinetics of the enzyme and the redox state of the active site metal. The Gln143 mutants were not product inhibited but were slower than the wild type by two to three orders of magnitude, and mutations at this site altered the redox state of the enzyme.;The midpoint potential of human Mn-SOD was measured both through single point equilibrium and redox titration. The two methods yielded agreeable values with Em = 393 +/- 35 mV. This value lies right between the reduction and oxidation midpoint potentials of superoxide, which facilitates both reactions.;Therefore, Gln143 and His30 are required for rapid catalysis but not essential for activity. The Gln143 stabilizes manganese in the oxidized state and contributes to the fine-tuning of the redox potential which is required for efficient catalysis. The His30 is involved in the formation of the product-inhibited complex in wild type human MnSOD; this inhibition is abolished in the mutant His30Asn. Therefore, this mutant is a good candidate for gene therapy research to provide better cytoprotection under states of oxidative stress.