Modulation of the cytochrome c oxidase activity by ATP: Implications for mitochondrial respiratory control

ATP and ADP are potential regulators of mitochondrial respiration and at physiological concentrations they affect the rate of electron transfer between cytochrome c and cytochrome c oxidase. The electron transfer, however, depends on the electrostatic interaction between the two proteins. In order to exclude any nonspecific ionic effects by these polyvalent nucleotides, we used 2'-O-(2,4,6)trinitro(TNP)-derivatives of ATP and ADP which have three orders of magnitude higher affinity for cytochrome c oxidase. A simple titration of the fluorescence intensity of TNP by cytochrome c oxidase showed a binding stoichiometry of 2:1 cytochrome c:cytochrome c oxidase. Higher ionic strength was required for TNP-ATP than for TNP-ADP to be dissociated from cytochrome c oxidase, indicating that the negative charges on the phosphate group are at least partially responsible for the binding. In both spectrophotometric and polarographic assays, addition of ATP (and ADP to a less extent) showed an enhanced cytochrome c oxidase activity. Both electron paramagnetic resonance and fluorescence spectra indicate that there is no significant change in the cytochrome c-cytochrome c oxidase interaction. Instead, reduction levels of the cytochromes at steady-state suggest that the increased activity of nucleotide-bound cytochrome c oxidase is due to faster electron transfer from cytochrome a to cytochrome aa which is known to be the rate limiting step in the oxygen reduction by cytochrome c oxidase.