Effect of Changing the Detergent Bound to Bovine Cytochrome c Oxidase upon Its Individual Electron-Transfer Steps

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.