Free -SH variations during ATP synthesis by oxidative phosphorylation in heart muscle mitochondria

Abstract

To investigate the possible role of thiols in the coupling mechanism, free −SH variations have been estimated along with ATP synthesis during oxidative phosphorylation in heart mitochondria. 1. 1. In well-coupled mitochondria, State 4 free −SH values amount to 43 nmoles/mg protein. 2. 2. The addition of ADP + P i , which initiates State 3 of oxidation and ATP synthesis, provokes an increase of free −SH (30% of State 4 values). If ADP or P i is added alone, no increase of free −SH occurs. 3. 3. m-Chlorocarbonyl cyanide phenylhydrazone prevents any −SH release upon addition of ADP + P i ; moreover, free −SH levels decrease in mitochondria while the respiratory chain is stimulated by the uncoupling agent. 4. 4. N-Ethyl maleimide added to well-coupled mitochondria blocks free −SH normally found in State 4 and strongly inhibits the respiratory level. Upon further addition of ADP, −SH release and stimulation of respiratory level as well as ATP synthesis are totally suppressed. 5. 5. Oligomycin does not affect the free −SH level in State 4 of oxidation but suppresses totally the −SH increase due to ADP + P i as well as ATP synthesis linked to respiratory chain oxidations. 6. 6. If cholate is added after the incubations and just before the 5,5'-dithiobis-(2-nitrobenzoic acid) estimation to eliminate membrane phenomena, the number of −SH evaluated are not significantly different and the increase observed in State 3 remains the same. These results are very much in favour of the participation of −SH with processes related to the coupling mechanism, since the −SH increase upon addition of ADP + P i cannot be related to changes in the redox state of the respiratory chain or the phosphate entry.