FLUOROALUMINUM AND FLUOROBERYLLIUM NUCLEOSIDE DIPHOSPHATE COMPLEXES AS PROBES OF THE ENZYMATIC MECHANISM OF THE MITOCHONDRIAL F1-ATPASE

The mechanism by which fluoride and aluminum or beryllium in combination with ADP inhibit beef heart mitochondrial F1-ATPase was investigated. The kinetics of inhibition depended on the nature of the anion present in the F1-ATPase assay medium. Inhibition required the presence of Mg2+ and developed more rapidly with sulfite and sulfate than with chloride, i.e., with anions which activate F1-ATPase activity. The ADP-fluorometal complexes were bound quasi-irreversibly to F1, and each mole of the inhibitory nucleotide-fluorometal complex was tightly associated with 1 mol of Mg2+. One mole of nucleotide-fluorometal complex was able to inhibit the activity of 1 mol of catalytic site in F1. Direct measurements of bound fluoride, aluminum, beryllium, and ADP indicated that the F1-bound ADP-fluorometal complexes are of the following types: ADP1Al1F4, ADP1Be1F1, ADP1Be1F2, or ADP1Be1F3. Fluoroaluminates or fluoroberyllates are isomorphous to P(i), and the inhibitory nucleotide-fluorometal complexes mimicked transient intermediates of nucleotides that appeared in the course of ATP hydrolysis. On the other hand, each mole offully inhibited F1 retained 2 mol of inhibitory complexes. The same stoichiometry was observed when ADP was replaced by GDP, a nucleotide which, unlike ADP, binds only to the catalytic sites of F1. These results are discussed in terms of stochastic model in which three cooperative catalytic sites of F1 function in interactive pairs.