ASSEMBLY OF A MEMBRANE-BOUND MULTI-ENZYME COMPLEX

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The formate-nitrate reductase pathway in Escherichia coli is being employed as an experimental system for studying the biogenesis of an organized, membrane-bound multienzyme complex. The formate-nitrate reductase pathway in E. coli is an electron transport system which is organized in the cell membrane so that energy, in the form of a protein gradient, is generated during transfer of electrons from formate to nitrate. Our long range goal is to define the molecular mechanisms in the biosynthesis of the component proteins, their insertion into the membrane, their assembly into multisubunit complexes and the incorporation of non-protein cofactor components into the functional enzyme complexes. Our immediate specific aims are focused on one of the enzyme complexes of this pathway, nitrate reductase; We propose to utilize biochemical, genetic and recombinant DNA techniques to determine (1) the structure of the nar operon and the specific component involved in its regulation, (2) the products of the nar operon, including the enzyme subunits and other possible regulatory components, (3) the role of specific subunits in the interaction of the enzyme with the membrane both in vitro and in vivo. Longer range studies will initiate studies on the role of specific protein domains on subunit interactions and insertion into the membrane.