Our research is concerned primarily with the role of protein-lipid and protein-protein interactions within membranes. We are studying components of the E. coli aerobic respiratory chain as a model of a multi-protein, membrane-bound enzyme network. Emphasis is on two purified components, pyruvate oxidase, and the cytochrome b/d terminal oxidase. Pyruvate oxidase is a flavoprotein dehydrogenase. We have characterized the lipid-binding properties of this protein, as well as the kinetic activation by lipids, the kinetic mechanism, and the nature of the active site. We now plan to characterize how this enzyme couples to other components in the E. coli membrane, and to obtain the complete amino acid primary sequence. The latter goal will be accomplished by cloning and sequencing the DNA which codes for the enzyme. Crystallographic studies to obtain the three-dimensional structure of the enzyme will be initiated. These studies offer the exciting prospect of yielding high resolution structural information of a lipid-requiring membrane enzyme. Our studies over the last three years have resulted in opening up the E. coli respiratory chain to detailed biochemical and genetic studies. The respiratory chain contains two branches and a total of 5 or 6 cytochromes. We recently purified a complex which contains half of the E. coli cytochromes and functions as a ubiquinol oxidase. This essentially is one of the two branches in the respiratory chain. This enzyme will be structurally and functionally characterized both in the E. coli membrane and in its purified form. Evidence indicates that proton translocation across the bilayer accompanies electron transfer through this complex in the E. coli membrane. Hence, the role of the cytochrome b/d complex in E. coli bioenergetics will be examined on a molecular level. Reconstitution studies in liposomes with pyruvate oxidase and other E. coli dehydrogenases along with the cytochrome b/d complex will be carried out and the results compared with those obtained in E. coli membrane preparations.
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