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 spectroscopic 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 proposed research offers a fresh approach to the complex problem of iron-sulfur cluster characterization, by way of a novel spectroscopic technique, variable-temperature magnetic circular dichroism. This technique affords an unambiguous method for identifying the cluster type of individual chromophoric centers in multicomponent enzymes. Furthermore, electronic and magnetic ground state information may be assessed by monitoring the detailed magnetic field and temperature dependence of individual optical transitions. The proposed development of an optical-microwave double resonance spectrometer will facilitate accurate optical determination of ground state g-factors, providing an invaluable link between optical and electron paramagnetic resonance transitions. The results will resolve numerous controversies and ambiguities in electron paramagnetic resonance and core extrusion data concerning the number, type and diversity of iron-sulfur centers in mitochondrial enzymes.
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