8-Azido-ATP modification of cytochrome c: retardation of its electron-transfer activity to cytochrome c oxidase.
Horse heart cytochrome c has been modified by 8-azido-ATP and the electron-transfer activity of the modified cytochrome c's to bovine heart cytochrome c oxidase (CcO) under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The intermolecular electron transfer between the redox protein partners was shown to be extremely slow. The 8-azido-ATP-modified system exhibited less than 5% of the intracomplex electron-transfer rate observed between native cytochrome c and CcO under otherwise identical conditions. The binding affinity of the modified cytochrome c was greatly reduced (3 orders of magnitude) at low ionic strengths; however, it was only slightly reduced (by a factor of 2) relative to the native protein at physiological ionic strengths. Thus, the binding affinity of the ATP-cytochrome c adducts is relatively insensitive to the ionic strength compared to the native enzyme, suggesting that a different docking conformation is assumed by the ATP-cytochrome c adducts in their interaction with the oxidase. Since the redox potential of the modified cytochrome c is close to the value of its native form, we conclude that there has been a change in the docking of the cytochrome c to CcO and the electronic coupling between heme c and CuA upon 8-azido-ATP modification.
Lin
J
J
A.A. Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125.
Wu
S
S
Lau
W T
WT
Chan
S I
SI
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000737:chemistry*; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006736:Horses; D066298:In Vitro Techniques; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism; D010084:Oxidation-Reduction; D010782:Photolysis
D001386:Azides; D003574:Cytochrome c Group; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV