Effect of neutral and acidic phospholipids on mitochondrial ATP synthase secondary structure.

The secondary structure of delipidated and egg phosphatidylcholine or asolectin reconstituted mitochondrial ATP synthase complex from beef heart was investigated by Fourier transform infrared spectroscopy. Upon reconstitution, the infrared spectra of ATP synthase revealed an increase in turns and a concomitant decrease in beta-sheet content which occurred to a larger extent in the presence of asolectin rather than in the presence of egg phosphatidylcholine. These data correlate with kinetic data showing a higher ATPase activity of the asolectin reconstituted enzyme protein than the egg phosphatidylcholine reconstituted or delipidated enzyme complexes.

H2O2-induced damage to beef heart mitochondria F0F1 ATP synthase complex: differential sensitivity of the F1 and F0 moieties.

Exposure of purified mitochondrial F0F1 ATP synthase to H2O2 resulted in a marked inhibition of the ATPase activity, irrespective of the purification procedure used and of the incorporation of the enzyme complex into phospholipid vesicles. The inactivation appeared consequent to oxidative modifications of the F1 moiety, whereas damage to the F0 sector, leading to low enzyme activity through impaired binding with F1, seemed not to occur. In fact, when H2O2-inactivated complex was deprived of F1, no loss of the capacity of the F0 sector thus obtained to properly reassemble with untreated purified F1 was apparent, because the resulting enzyme complex showed full activity and oligomycin sensitivity. On the contrary, the exposure of the isolated components F1 or F0 to H2O2, followed by reassembly with untreated F0 and F1 respectively, resulted in both cases in lower catalytic activity of the reconstituted complexes, whereas low oligomycin sensitivity was exhibited only in the case of F0 treatment, suggesting the inactivation in this case as due to oxidative modifications leading to impaired binding with F1.

The inactivation of mitochondrial F1 ATPase by H2O2 is mediated by iron ions not tightly bound in the protein.

Exposure to purified mitochondrial F1 ATPase to continuous flux of H2O2 resulted in significant loss (up to 60%) of the ATP hydrolytic activity. The presence of chelating agents including desferrioxamine or previous selective removal of the iron ions not tightly bound in the protein completely prevented the inactivation, whereas re-loading of the enzyme with F3+ restored the sensitivity to H2O2. A marked protective effect was provided as well by mannitol or by Cu,Zn superoxide dismutase. The results indicated the decomposition of H2O2 by redox-active iron-protein adducts as responsible for the enzyme inactivation, probably through site-directed generation of more highly reactive oxygen species. A possible role for iron associated to F1 component in the oxidation, aging and turnover of ATP synthase complex in vivo may be suggested on the basis on these results.