Investigation of the Maturation of Yeast Superoxide Dismutase: Effect of the Copper Chaperone, the Disulfide Bond, and Metal Binding

Superoxide is formed by the one electron reduction of dioxygen. Superoxide damages cells directly, and further reduction of superoxide produces other reactive molecules that can damage cells. Superoxide dismutase enzymes defend against superoxide by catalyzing the disproportionation of two superoxide molecules to hydrogen peroxide and dioxygen.;In this dissertation, copper-zinc superoxide dismutase (Sod1) is examined. By various measurements of Sod1 activity we found Sod1 from the budding yeast Saccharomyces cerevisiae is active, obtains metal in vivo , and has in oxidized disulfide bond, all in the absence of the copper chaperone for Sod1 (CCS). We also found yeast Sod1 is active in the absence of the intrasubunit disulfide bond in Sod1, but activity is lower than wild type Sod1, possibly due to lower ionic attraction for superoxide. The findings of activity without CCS and activity in the absence of the disulfide bond both contradicted earlier work.;We also explored metal binding to yeast and tomato Sod1. We found that metal binding to the zinc site of yeast Sod1 at pH 5.5 is impaired in the absence of the disulfide bond, but the addition of copper or an increase in pH restores binding at the zinc site. We found tomato Sod1 does not bind metals below pH 5.0, corroborating the finding of our collaborator who showed an impaired zinc binding site in a crystal structure at pH 4.6.;Finally, we designed and synthesized, up to the final step, a molecular wire for insertion into the active site of Sod1. The wire is to be used to connect Sod1 to an electrode for determination of the reduction potential of the copper in Sod1.