The research proposed is intended to expand knowledge on how iron-sulfer clusters and covalent flavins assemble and function in membrane-bound protein complexes required for electron transport and energy conservation. The studies are aimed at answering the existing questions relating to the interrelationship between the three iron-sulfur clusters known to be present in fumarate reductase and whether there is a requirement for a "flavin-ligating" enzyme to catalyze the formation of covalent flavin protein complexes. The research will also address questions about the relationship between iron-sulfur cluster structure and protein sequence and the role of the individual clusters in electron transport. Approaches used to meet these goals include genetic modification of fumarate reductase by both oligonucleotide-directed and chemical mutagenesis methods. The modified proteins will then be analyzed by biochemical assays in order to define the catalytic activity of the modified enzyme and its cellular location (membrane-bound or cytoplasmic). In addition we will use biophysical approaches such as EPR and magnetic circular dichroism to monitor the function and structure of the iron-sulfur clusters of the enzyme. This research will contribute to our understanding of how complex flavoprotein oxidoreductases assemble into functional protein complexes and thus help elucidate the path of electron transport through such proteins.
