DOES PYROPHOSPHATE BIND TO THE CATALYTIC SITES OF MITOCHONDRIAL F1-ATPASE

The interactions between the pyrophosphate (PP(i)) binding sites and the nucleotide binding sites on mitochondrial F1-ATPase have been investigated, using F1 preparations containing different numbers of catalytic and noncatalytic nucleotide-binding sites occupied by ligands. In all cases, the total number of moles of bound nucleotides and PP(i) per mole of F1 was less than or equal to six. F1 preparations containing either three or two filled noncatalytic sites and no filled catalytic sites (referred as F1[3,0] and F1[2,0]) were found to bind 3 mol of PP(i)/mol of F1. Tight binding of ADP-fluoroberyllate complexes to two of the catalytic sites of F1 converted the three heterogeneous PP(i)-binding sites into three homogeneous binding sites, each exhibiting the same affinity for PP(i). The addition of PP(i) at saturating concentrations to F1 containing GDP bound to two catalytic sites (F1[2,2]) resulted in the release of 1 mol of GDP. Furthermore, the addition of PP(i) to F1 filled with ADP-fluoroberyllate at the catalytic sites resulted in the release of 1 mol of tightly bound ADP/mol of F1. Taken together, these results indicate that PP(i) binds to specific sites that interact with both the catalytic and the noncatalytic nucleotide-binding sites of F1.