Electron transfer from cytochrome c to 8-azido-ATP-modified cytochrome c oxidase.
Bovine heart cytochrome c oxidase (CcO) has been modified by 8-azido-adenosine 5'-triphosphate (8-azido-ATP), and the electron-transfer activity from ferrocytochrome c to the modified CcO under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The kinetics of intermolecular electron transfer between the redox protein partners was shown to be reduced significantly. In addition, there is significant decrease in the binding affinity of the cytochrome c to the oxidase upon 8-azido-ATP modification. The 8-azido-ATP-modified CcO exhibited 50% of the intracomplex electron-transfer rate (ket) and 56% of the association constant (Ka) normally observed between cytochrome c and native CcO under otherwise identical conditions. Since the effective electron transfer rate constant is the product of ket and Ka under nonsaturation conditions, the overall electron-transfer rate has been curtailed by over a factor of 2. Similar observations have been noted with the native CcO in the presence of 3 mM ATP. In contrast, the redox potential of neither CuA nor cytochrome a was altered upon 8-azido-ATP modification or in the presence of 3 mM ATP. Also, no gross structural changes at either the CuA or the cytochrome a site were noted, as evidenced by a lack of any spectral perturbations in the EPR signals from both of these centers. We conclude that ATP modulates the electron transfer from cytochrome c to CcO by interacting with the CcO and altering allosterically the docking. In this manner, ATP can affect the branching of the electron input from ferrocytochrome c to cytochrome a and CuA.
Lin
J
J
A. A. Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125, USA.
Wu
S
S
Chan
S I
SI
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000737:chemistry*; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding
D000345:Affinity Labels; D001386:Azides; D003574:Cytochrome c Group; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV