Characterization of steady-state activities of cytochrome c oxidase at alkaline pH: mimicking the effect of K-channel mutations in the bovine enzyme.
The cytochrome c oxidase activity of the bovine heart enzyme decreases substantially at alkaline pH, from 650 s(-1) at pH 7.0 to less than 10 s(-1) at pH 9.75. In contrast, the cytochrome c peroxidase activity of the enzyme shows little or no pH dependence (30-50 s(-1)) at pH values greater than 8.5. Under the conditions employed, it is demonstrated that the dramatic decrease in oxidase activity at pH 9.75 is fully reversible and not due to a major alkaline-induced conformational change in the enzyme. Furthermore, the Km values for cytochrome c interaction with the enzyme were also not significantly different at pH 7.8 and pH 9.75, suggesting that the pH dependence of the activity is not due to an altered interaction with cytochrome c at alkaline pH. However, at alkaline pH, the steady-state reduction level of the hemes increased, consistent with a slower rate of electron transfer from heme a to heme a3 at alkaline pH. Since it is well established that the rate of electron transfer from heme a to heme a3 is proton-coupled, it is reasonable to postulate that at alkaline pH, proton uptake becomes rate-limiting. The fact that this is not observed when hydrogen peroxide is used as a substrate in place of O2 suggests that the rate-limiting step is proton uptake via the K-channel associated with the reduction of the heme a3/CuB center prior to the reaction with O2. This step is not required for the reaction with H2O2, as shown previously in the examination of mutants of bacterial oxidases in which the K-channel was blocked. It is concluded that at pH values near 10, the delivery of protons via the K-channel becomes the rate-limiting step in the catalytic cycle with O2, so that the behavior of the bovine enzyme resembles that of the K-channel mutants in the bacterial enzymes.
Riegler
David
D
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
Shroyer
Lois
L
Pokalsky
Christine
C
Zaslavsky
Dmitry
D
Gennis
Robert
R
Prochaska
Lawrence J
LJ
D000818:Animals; D002417:Cattle / Q000378:metabolism*; D004579:Electron Transport / Q000502:physiology; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D015221:Potassium Channels / Q000378:metabolism*; D013057:Spectrum Analysis
D015221:Potassium Channels; D006418:Heme; D006861:Hydrogen Peroxide; D003576:Electron Transport Complex IV