A genetic and biochemical analysis of the structure of subunit A and its role in proton translocation by the F(1)F(0) ATP synthase of Escherichia coli

The membrane bound F0 sector of the Escherichia coli F1F0 ATP synthase consists of three subunits present in a a1b2c12 stoichiometry. My thesis research has focused on the a subunit of the F 0 sector. The transmembrane topography of the a subunit and the role of the Arg210 and Glu219 residues of subunit a in proton translocation by the F0 sector have been investigated. The topology of subunit a was determined from chemical modification studies of genetically introduced cysteine residues. Subunit a was shown to consist of five transmembrane helices with the N-terminus as periplasmic and the C-terminus as cytoplasmic. Possible interactions between the transmembrane segments are suggested by second site suppressor mutations that have been isolated. The topological data when combined with data obtained from the second site suppressor mutations enables the generation of a model for the folding of subunit a in the membrane. Site directed mutagenesis has been used to explore the requirement of Arg210 and Glu219 of subunit a for proton translocation through the F 0 sector. The mutational analysis demonstrates that the Arg210 and Glu219 residues of subunit a are not absolutely required for proton translocation through the F0 sector.;Studies on the modification of Asp61 of subunit c of the F0 sector by dicyclohexylcarbodiimide (DCCD) have also been carried out. Asp61 of the c subunit is the key proton binding site during proton translocation through F0 and can be specifically modified by DCCD. Based upon the pH dependence of the reaction of DCCD with Asp61, the pKa of Asp61 has been determined to be around 8.0. The pH dependence of the DCCD reaction is not seen with the membrane bound enzyme. The lack of a pH dependence for the DCCD modification reaction with the membrane bound enzyme is in accord with the placement of the Asp61 residue of subunit c in the middle of the lipid bilayer for the native F1F 0 complex.