ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway

Abstract

Eukaryotic cytochrome c possesses an ATP-binding site of substantial specificity and high affinity that is conserved between highly divergent species and which includes the invariant residue arginine''. Such evolutionary conservatism strongly suggests a physiological role for ATP binding that demands further investigation. We report the preparation of adducts of the protein and the affinity labels 8-azido adenosine 5'-triphosphate, adenosine S-triphosphate-T,3'-diaIdehyde, and 5'-p-fluorosulfonylbenzoyladenosine. The two former reagents were seen to react at the arginine9'-containing site, yet the reaction of the latter, although specific, occurred elsewhere, suggesting caution is necessary in its use. None of the adducts displayed significant modification of global structure, stability, or physicochemical properties, leading us to believe that the 8-N3-ATP and oATP adducts are good stabilized models of the noncovalent interaction; yet modification led to significant, and sometimes pronounced, effects on biological activity. We therefore propose that the role of ATP binding to this site, which we have shown to occur when the phosphorylation potential of the system is high under the equivalent of physiological conditions, is to cause a decrease in electron flow through the mitochondrial electron transport chain. Differences in the degree of inhibition produced by differences in adduct chemistry suggest that this putative regulatory role is mediated primarily by electrostatic effects.

ATP Induces a Conformational Change in Lipid-bound Cytochrome c

“…A high affinity binding site for ATP has been described and shown to involve the invariant Arg 91 (5,(7)(8)(9)(10). The involvement of Arg 91 in binding of ATP and consequent modulation of electron transfer by the protein has been investigated previously by semisynthetic analogs of cyt c in which this single arginine residue of the 66 -104 peptide was chemically modified by cyclohexane-1,2-dione prior to ligation with the 1-65 peptide (10).…”

“…In cyt c part of the high affinity ATP-binding site is constituted by the invariant Arg 91 (5,(7)(8)(9)(10), and binding of ATP to this decreases the rate of electron flow through the mitochondrial electron transport chain (9). Accordingly, when the ATP-binding site of cyt c is occupied by a covalently bound ATP, the electron-transfer activity of cyt c with reductase and oxidase are inhibited to 41 and 11-15%, respectively, of the values measured for the native protein (3).…”

The effects of ATP and sodium chloride on the cytochrome c–cardiolipin interaction: The contrasting behavior of the horse heart and yeast proteins

“…Previous studies have shown that, contrary to yeast, horse cyt c possesses (at least) one binding site with high affinity for ATP [18,19,37]. This is correlated with the critical role played by cyt c in cell apoptosis.…”

Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts

“…It has been shown that anions directly bind cytochrome c around the polylysine binding pocket [13,14]. This binding of ATP may be physiologically important for feedback inhibition of the respiratory chain [15].…”