Effect of diethyl pyrocarbonate modification on spectral and steady-state kinetic properties of bovine heart cytochrome oxidase.
The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and alpha bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85-90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.
Doran
J D
JD
Department of Biological Sciences, University of Calgary, Alta., Canada.
Hill
B C
BC
D000818:Animals; D002417:Cattle; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006639:Histidine; D007700:Kinetics; D008081:Liposomes; D008954:Models, Biological; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D011522:Protons; D013056:Spectrophotometry, Ultraviolet
D008081:Liposomes; D011522:Protons; D006639:Histidine; D003576:Electron Transport Complex IV; D004047:Diethyl Pyrocarbonate