Effect of changing the detergent bound to bovine cytochrome c oxidase upon its individual electron-transfer steps

• Published in: Biochemistry (Easton) Vol. 29; no. 3; pp. 764 - 770
• Main Authors: Mahapatro, Surendra N, Robinson, Neal C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 23.01.1990
• Subjects: Aerobiosis; Animals; Artificial membranes and reconstituted systems; Biological and medical sciences; Cattle; Cyanides - metabolism; detergents; Detergents - pharmacology; electron transfer; Electron Transport - drug effects; Electron Transport Complex IV - metabolism; Fundamental and applied biological sciences. Psychology; Horses; Kinetics; Membrane physicochemistry; Molecular biophysics; Oxidation-Reduction; Oxygen - metabolism; Protein Binding; Surface-Active Agents - pharmacology; Cytochrome c oxidase; Detergent; Bovine; Enzyme; Electron transfer; Artificial membrane; Vertebrata; Mammalia; Enzymatic activity; Artiodactyla; Ungulata; Conformation; Ultraviolet visible spectrometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00455a025

The influence of the detergent environment upon individual electron-transfer rates of cytochrome c oxidase was investigated by stopped-flow spectrophotometry. The effects of three detergents were studied: lauryl maltoside, which supports a high turnover number (TN = 350 s-1), n-dodecyl octaethylene glycol monoether (C12E8), which supports an intermediate TN (150 s-1), and Triton X-100 in which oxidase is nearly inactive (TN = 2-3 s-1). Under limited turnover conditions (cytochrome c:cytochrome c oxidase ratio = 1:1 to 8:1), the rate of oxidation of cytochrome c was measured and compared with the fast reduction of cytochrome a and its relatively slow reoxidation. Two reducing equivalents of cytochrome c were rapidly oxidized in a burst phase; the remaining two to six equivalents were oxidized more slowly, concurrent with the reoxidation of cytochrome a; i.e., the percent reduced cytochrome a reflects the percent reduced cytochrome c. With the resting enzyme, the bimolecular reaction between reduced cytochrome c and cytochrome a was rapid, was insensitive to the detergent environment, and was not the rate-limiting step in the presence of any detergent. The rate of internal electron transfer from cytochrome a to cytochrome a3 in the resting enzyme was slow and only slightly affected by the detergent environment: 1.0-1.1 s-1 in Triton X-100, 5-7 s-1 in C12E8, and 5-12 s-1 in lauryl maltoside. With the pulsed enzyme, the intramolecular electron transfer between cytochrome a and cytochrome a3 increased 4-5-fold in the lauryl maltoside enzyme but did not increase in the Triton X-100 enzyme (intermediate values were obtained with the C12E8 enzyme). We conclude that cytochrome c oxidase acquires the pulsed conformation only in those detergents that support high TN's, e.g., lauryl maltoside and C12E8, but it is locked in the resting conformation in those detergents which result in low TN's, e.g., Triton X-100.