Studies of 8-azido-ATP adducts reveal two mechanisms by which ATP binding to cytochrome c could inhibit respiration.

We have proposed that the binding of ATP at a site of substantial affinity and specificity could regulate the activity of cytochrome c with its physiological partners and thus the overall efficiency of mitochondrial electron transport. We now describe the use of ATP affinity-labeled protein to test the effect of occupancy of that site, which includes the invariant arginine 91, on the activity of cytochrome c with purified cytochrome c reductase and oxidase and its association with the mitochondrial inner membrane. Electron-transfer activities with the reductase and oxidase were inhibited by site occupancy to 41% and 11-15% of native values, respectively. The marked difference in the degree of inhibition of activity that distinguishes the reactions with the two major physiological partners was sufficient to cause, in whole mitochondria, a demonstrable shift from a situation in which there is a rate-limiting transfer from the reductase to cytochrome c, to a state where rates are more evenly matched for transfers between cytochrome c and the two redox partners. Site occupancy also substantially reduces the ionic strength necessary for half-maximal dissociation of cytochrome c from the membrane. These data imply that the decreased efficiency of electron transfer caused by ATP attachment can be attributed to a decrease in the protein's activity with individual physiological partners, possibly compounded with a decrease in its affinity for the inner mitochondrial membrane, and suggest that feedback regulation by ATP of cellular respiration operates in like manner.