MOLECULAR GENETICS OF FLAVIN AND FE-S CONTAINING ENZYMES

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The objective of this research project is to gain a clearer understanding of the structure and function of flavin and [Fe-S] containing enzymes that participate in electron transfer processes in both eucaryotic and procaryotic organisms. Major efforts center on the bacterial model systems including the fumarate reductase enzyme complex (Frd) and the succinate dehydrogenase enzyme complex (Sdh) of Escherichia coli. The two bacterial enzymes are powerful models to examine questions regarding how this class of enzymes inter-convert fumarate and succinate, and function in concert with the cellular electron transport machinery to accomplish ATP synthesis via oxidative phosphorylation processes. The bacterial enzymes are remarkably similar structurally although each is uniquely different with respect to their prosthetic group redox properties and the type of quinone used. Since the molecular and biochemical tools for the Escherichia coli Frd and Sdh systems are extremely well developed, their continued application should aid in elucidating the assembly and structure of this class of membrane bound oxidoreductase complexes that contain covalently linked flavin and multiple non-heme iron centers. We will evaluate the roles of specific amino acids in providing structaure and function at the [2Fe-2S] center, the [3Fe-4S] center, and the [4Fe-4S] centers of FrdB, the active site of FrdA, and the quinone interaction sites in the membrane association FrdCD subunits. Predictions from these studies will be evaluated with the Sdh complex of E. coli. Chimeric Frd and Sdh complexes will be generated by use of protein domain swapping methods to test whether determinants referring specific enzyme properties can be localized. Knowledge gained from these studies should provide a detailed molecular description of the structure and function of this class of membrane bound enzymes. We are using a non-animal system to perform basic research in a cost effective and timely manner to address human problems for which no equivalent experimental system has yet been development.