Heat treatment of cytochrome c oxidase perturbs the CuA site and affects proton pumping behavior.
It has been previously reported that mild heat treatment (43 degrees C for ca. 60 min) abolishes the proton pumping activity of cytochrome c oxidase while leaving the oxidase activity and cytochromes a and a3 unperturbed [Sone, N., & Nicholls, P. (1984) Biochemistry 23, 6550-6554]. We herein describe the effects of this heat treatment on the electron paramagnetic resonance (EPR) and optical absorption signatures of the redox-active metal centers in the enzyme. We find that heat treatment of the oxidized enzyme causes a local structural perturbation at the CuA site. After heat treatment, the enzyme sample contains three subpopulations, each of which has a different structure at CuA. These include (i) native CuA, (ii) a type 2 copper species similar to the one produced by chemical modification by p-(hydroxymercuri)benzoate (pHMB) [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972], and (iii) a novel type 1 copper species. In addition to changes at the CuA site, we find that heat treatment results in accelerated cyanide binding and the removal of subunit III. If the cytochrome c oxidase is heat treated while fully reduced, none of these changes are observed except for subunit III depletion. Furthermore, partial (CO mixed-valence derivative) reduction of the enzyme as well as ligand binding to cytochrome a3 also protects the enzyme against the heat-induced changes, indicating that the oxygen binding site plays a role in stabilizing the CuA site against structural perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
P M
PM
Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125.
Morgan
J E
JE
Nilsson
T
T
Ma
M
M
Chan
S I
SI
D001665:Binding Sites; D002850:Chromatography, Gel; D003300:Copper / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000378:metabolism; D011522:Protons; D013053:Spectrophotometry
D003574:Cytochrome c Group; D007473:Ion Channels; D011522:Protons; D003300:Copper; D003576:Electron Transport Complex IV; D011190:Potassium Cyanide