ATP synthesis by oxidative phosphorylation is catalyzed by F0F1-ATP synthases found embedded in mitochondrial and bacterial membranes. The F1 portion of the complex contains the catalytic sites, can be readily solubilized, and has a subunit stoichiometry of alpha3-beta3-gamma-delta- epsilon. The structure, mechanism, and regulation of F1 is the subject of this proposal. F1 contains three catalytic and three noncatalytic nucleotide-binding sites. A topological model for the sites has been constructed based on affinity labeling, mutagenesis, predictions of secondary structure, and comparisons with proteins of known structure. The model places sites at or near alpha/beta interfaces, where a pair of catalytic and noncatalytic sites are oriented like the ATP and AMP sites of adenylate kinase. Residues predicted to interact with bound nucleotides will be altered by site-specific mutagenesis of the E. coli enzyme. The role of each will be assessed by kinetic analysis and nucleotide binding measurements. In some cases, where residues in the putative adenine-binding subdomains are changed to tyrosine, it may be possible to obtain a direct confirmation of their presence by testing for reactivity towards 2-azido-ATP. Similarly, when substituting lysine for residues thought to interact with phosphoryl groups, reactivity towards PLP-AMP, PLP-ADP, and PLP-ATP will be tested. Primary mutations that allow assembly of the synthase but give the unc- phenotype (low or no growth on succinate) will be used to select for second-site suppressor mutations. A targeted approach for random mutagenesis will employ double-stranded cassettes for small regions and a modified polymerase chain reaction for larger segments. Particular emphasis will be given to the identification of interacting sites between the alpha and beta subunits. Novel bifunctional probes will be used to further examine the orientation of sites. Bis-(2-azido)APxA will be tested for its ability to crosslink tyrosine that are known to be present at catalytic and noncatalytic sites. PLP-(2-azido)ADP will be used to establish the location of an alpha- subunit lysyl residue. Experiments are designed to determine whether noncatalytic site-bound nucleotides can act as acid catalysts or participate in the slow transfer of phosphoryl groups to and from catalytic sites. Further studies of the mechanism will include attempts to determine whether the three catalytic sites turn over in a sequential manner and whether rotary motion is obligatory in coupled reactions. Improvements in the kinetic analysis of mutant EcF1 will include determining conditions that avoid inhibition by the epsilon-subunit and by MgADP.
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