Respiration is the most fundamental of all life processes and detailed knowledge is essential to our understanding and treatment of numerous health related problems. The long-term goal of this proposal is a molecular understanding of the mitochondrial respiratory chain, with particular emphasis on electron transport and redox-linked proton translocation. Iron- sulfur clusters are ubiquitous in mitochondrial electron transport and intimate knowledge of their type and function is a necessary prerequisite in the mechanistic elucidation of both processes. The primary objectives of this proposal are the identification of the functional, structural, electronic and magnetic properties of the multiple iron-sulfur centers in beef heart mitochondria. This will be accomplished by investigations of the membrane-bound enzymes of the mitochondrial electron transport chain, NADH dehydrogenase, succinate dehydrogenase, electron transport flavoprotein dehydrogenase and the Rieske center as well as the soluble mitochondrial enzyme, aconitase. The approach is spectroscopic involving several mutually complementary techniques; notably variable temperature magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman. When coupled with systematic chemical change, these techniques provide a means of selectively investigating individual iron-sulfur clusters in multicomponent metalloenzymes. Parallel spectroscopic studies of a range of bacterial iron-sulfur enzymes and proteins are also proposed, e.g. ferredoxins, fumarate reductase from Escherichia coli, glutamine amidoribosyltransferase form Bacillus subtilis, nitrogenase Fe protein and FeV and FeMo proteins from Azotobacter vinelandii. These experiments will increase understanding of the functional and structural diversity of biological iron-sulfur clusters, and facilitate interpretation of results from mitochondrial enzymes. In addition they address questions of fundamental importance in the area of biological nitrogen fixation.
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