Labeling of thiols involved in the activity of complex V of the mitochondrial oxidative phosphorylation system.

The effects of various thiol reagents on the oligomycin-sensitive ATPase and ATP-Pi exchange activities of Complex V (ATP synthetase complex) purified according to Stiggall, D. L., Galante, Y. M., and Hatefi, Y.((1978) J. BioL Chem 253,956-964) were studied.Parachloromercuribenzoate (p-CMB) completely inhibited both activities.Disulfide reagents, such as 5,5'-dithiobisdinitrobenzoate or carboxypyridine disulfide (CPDS), inhibited the ATP-Pi exchange activity but did not modify the ATPase activity.N-ethylmalemide (2 111~) only slightly diminished the exchange activity of Complex V.Both the exchange and hydrolytic activities of Complex V treated with N-ethylmaleimide could be completely inhibited upon subsequent addition of p- CMB.Binding of radioactive CPDS to Complex V was completely prevented by p-CMB, which indicated that the thiol groups accessible to CPDS were also modified by pCMB.A comparison between the binding of ['4C]pCMB with or without protection by unlabeled Nethylmaleimide allowed the conclusion that, under assay conditions, p-CMB reaches thiols unreactive toward N-ethylmaleimide.The inhibitions by p-CMB or disulfide reagents could be reversed under certain conditions by cysteine or dithiothreitol.The location of the thiol groups involved in Complex V activity was studied in the following manner.1) Cold N-ethylmaleimide was added to Complex V to block thiols not involved in ATP-Pi exchange. 2)Sufficient CPDS or pCMB was added to bind and protect the thiols related to ATP-Pi exchange activity.Then, excess reagent was removed by repeated ethanol precipitations of Complex V. 3) Complex V was denatured by sodium dodecyl sulfate (SDS); then all of the newly exposed thiols were blocked by cold N-ethylmaleimide.4) An excess of dithiothreitol was added to regenerate the thiols previously protected by p-CMB or CPDS (dithiothreitol would be expected also to convert some disulfide bridges to new thiols). 5)After elimination of dithiothreitol by repeated precipitations of Complex V, ["GIN-ethylmaleimide was added to specifically label the thiols previously protected byp-CMB or CPDS, plus those generated from disulfides.