Environment of the sulfhydryl groups in bovine heart mitochondrial H+-ATPase

Electron transport particles and purified H+-ATPase (F1-F0) vesicles from beef heart mitochondria have been treated with two classes of thiol reagent, viz. membrane-impermeable organomercurials and a homologous series of N-polymethylene carboxymaleimides (Mal-(CH2)x-COOH or AMx). The effect of such treatment on ATP-driven reactions (ATP-Pi exchange and proton translocation) has been examined and compared to the effects on rates of ATP hydrolysis. The organomercurials inhibited ATP-Pi exchange and one of them (p-chloromercuribenzoate) inhibited ATPase activity. Of the maleimide series (AMx), AM10 and AM11 inhibited both ATP-Pi exchange and ATP-driven membrane potential, but not ATPase activity. The other members of the series were essentially inactive. N-Ethylmaleimide was intermediate in its efficacy. Passive H+ conductance through the membrane sector F0 was 50% blocked by AM10, slightly blocked by AM2 and N-ethylmaleimide, and unaffected by the other members of the AMx series. The data imply that one -SH near the membrane surface and one -SH about 12 A from the surface are functional in proton translocation through the H+-ATPase.