In a six-stage experimental design, we shall study, in detail, the relative locations and catalytic functions of the adenine nucleotide binding sites on the beef heart and rat liver mitochondrial F1-ATPase enzymes. The novelty of our approach uses new, site-directed photoaffinity labels synthesized by us, each possessing benzophenone as the photoactive functional group: BzATP/BzADP; BzAF (a fluorescent, nucleotide-site-selective probe); and Iodo-BzATP (a heavy atom modification of BzATP). 3H-, 32P- and 14C-derivatives of these photoaffinity probes offer the means of sensitive detection of subunit location and arginine-peptide mapping upon photolytically-induced covalent binding to F1, and, as well, the ability to assess low levels of catalytic activity with several of these ATP substrate analogs prior to photolysis. Both native and nucleotide-depleted BHF1 shall be examined with regard to the subunit location, binding stoichiometry, and catalytic characteristics of Bz-nucleotide covalent binding to (1) """"""""exchangeable"""""""" (i.e., catalytic) and (2) """"""""non-exchangeable"""""""" (i.e., presumably non-catalytically competent) sites/mol F1. Attempts to label, exclusively, the """"""""non-exchangeable"""""""" nucleotide sites will be performed, and, if successful, the effects on ATP turnover shall be assessed. The newly synthesized, pH-sensitive fluorescent probe, BzAF (a fluorescein derivative), shall be employed, for the first time, as a direct, site-specific monitor of potential conformational changes at the catalytic site(s) of F1 that may occur in response to shifts in the [H+] of the system. Determinations of fluorescence lifetimes and polarization of the bound fluorescein moiety can provide critical information on the immediate environment of the adenine nucleotide binding sites, and yield information on the possible environmental heterogeneity of such sites. Via a collaborative arrangement with L.M. Amzel, Iodo-Bz-nucleotide labeled rat liver F1 shall be subjected to crystallization and X-ray diffraction analysis in order to determine the topological locus of at least one of the catalytic site domains on this ubiquitous, truly life-supporting enzyme.
