Properties of bound inorganic phosphate on bovine mitochondrial F1F0-ATP synthase

Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (P-i). This phosphate exchanged completely with exogenous P-32(i) when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by P-32(i) when the enzyme was not pretreated with DMSO. These two molecules of P-32(i) were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound P-32(i) remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of P-32(i) were displaced by ATP. The ATP-resistant P-32(i) was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of P-32(i) to DMSO, followed by removal of the DMSO, resulted in the loss of the bound P-32(i) and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on thr enzyme was occupied by ATP, which could undergo a P-i <-> ATP exchange reaction with bound P-i The presence of two catalytic sites containing bound P-i is consistent with the X-ray crystallographic structure of F-1 (Bianchet, er at, 1998). Thus, five of the six molecules of bound P-i were accounted for. Three molecules of bound P-i were at catalytic sites and participated in ATP synthesis or P-i <-> ATP exchange. Two other molecules of bound P-i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P-i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers.