ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway.
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 arginine91. 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 5'-triphosphate-2',3'-dialdehyde, and 5'-p-fluorosulfonylbenzoyladenosine. The two former reagents were seen to react at the arginine91-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.
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
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Craig
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Wallace
D000241:Adenosine / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D066298:In Vitro Techniques; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D001386:Azides; D003574:Cytochrome c Group; C004282:periodate-oxidized adenosine 5'-triphosphate; C017040:8-azidoadenosine 5'-triphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D000241:Adenosine