Manganese metalloenzymes serve essential roles in biology ranging from antioxidant defense to oxygenic photosynthesis, the unique chemistry of each site being templated by its novel protein environment. The specialized catalytic motifs that direct and control the reactivity of the metal ion in these active sites are now emerging from biostructural studies. This proposal aims to combine advanced spectroscopy, mechanistic biochemistry, and structural biology to probe the fundamental patterns of reactivity defined by the catalytic motifs in four manganese metalloenzymes (Mn superoxide dismutase, Mn catalase, oxalate oxidase, and oxalate decarboxylase). These enzymes, all involved in fundamental cellular responses to environmental stress, represent both mononuclear and binuclear Mn active sites, and together illustrate key features of Mn redox biochemistry. Multiple approaches will be used to establish the underlying catalytic principles expressed in these manganese active sites. Site-directed mutagenesis of the structurally defined recombinant enzymes will dissect the functions of specific residues and correlate with rapid kinetic analysis of reactivity to experimentally define the role of catalytic elements in the metalloprotein complexes. Structures of important complexes will be solved at molecular resolution by X-ray crystallography, and at electronic resolution by spectroscopic methods (electronic absorption, CD, variable temperature MCD, FTIR and EPR polarization). These combined experimental approaches are expected to give new insight into the fundamental chemistry of manganese metalloenzyme complexes and provide the basis for a detailed understanding of the unique catalytic roles of manganese in biology.
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