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

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

Journal of Biological Chemistry 256(13): 6776-6782

The effects of various thiol reagents on the oligomycin-sensitive ATPase and ATP-Pi exchange activities of Complex V (ATP synthetase complex) [from pig or beef heart] were studied. p-Chloromercuribenzoate (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-ethylmaleimide (NEM) (2 mM) only slightly diminished the exchange activity of Complex V. Both the exchange and hydrolytic activities of Complex V treated with NEM 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 p-CMB. A comparison between the binding of [14C]p-CMB with or without protection by unlabeled NEM allowed the conclusion that, under assay conditions, p-CMB reaches thiols unreactive toward NEM. 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. Cold NEM was added to Complex V to block thiols not involved in ATP-Pi exchange. Sufficient CPDS or p-CMB 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. Complex V was denatured by sodium dodecyl sulfate (SDS); then all of the newly exposed thiols were blocked by cold NEM. 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). After elimination of dithiothreitol by repeated precipitations of Complex V, [14C]NEM was added to specifically label the thiols previously protected by p-CMB or CPDS, plus those generated from disulfides. The thiols which react with CPDS or p-CMB and which are involved in the ATP-Pi exchange activity of Complex V apparently are located at the level of low MW protein(s) (8-10 .times. 103) and at the level of the .alpha. subunit of the F1 component of Complex V.

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Accession: 005787089

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PMID: 6453870

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