Mahapatro S N, Robinson N C
Department of Biochemistry, University of Texas Health Science Center, San Antonio, 78284-7760.
Biochemistry. 1990 Jan 23;29(3):764-70. 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.
通过停流分光光度法研究了去污剂环境对细胞色素c氧化酶单个电子转移速率的影响。研究了三种去污剂的作用：月桂基麦芽糖苷，其支持高周转数（TN = 350 s⁻¹）；正十二烷基八乙烯二醇单醚（C12E8），其支持中等周转数（150 s⁻¹）；以及Triton X - 100，其中氧化酶几乎无活性（TN = 2 - 3 s⁻¹）。在有限周转条件下（细胞色素c：细胞色素c氧化酶比例 = 1:1至8:1），测量细胞色素c的氧化速率，并与细胞色素a的快速还原及其相对缓慢的再氧化进行比较。两个还原当量的细胞色素c在一个突发阶段迅速被氧化；其余两到六个当量氧化得更慢，与细胞色素a的再氧化同时发生；即，还原的细胞色素a的百分比反映了还原的细胞色素c的百分比。对于静止酶，还原的细胞色素c与细胞色素a之间的双分子反应迅速，对去污剂环境不敏感，并且在任何去污剂存在下都不是限速步骤。静止酶中从细胞色素a到细胞色素a3的内部电子转移速率缓慢，仅受去污剂环境轻微影响：在Triton X - 100中为1.0 - 1.1 s⁻¹，在C12E8中为5 - 7 s⁻¹，在月桂基麦芽糖苷中为5 - 12 s⁻¹。对于脉冲酶，细胞色素a与细胞色素a3之间的分子内电子转移在月桂基麦芽糖苷酶中增加了4 - 5倍，但在Triton X - 100酶中没有增加（C12E8酶得到中间值）。我们得出结论，细胞色素c氧化酶仅在那些支持高周转数的去污剂中获得脉冲构象，例如月桂基麦芽糖苷和C12E8，但在那些导致低周转数的去污剂中被锁定在静止构象，例如Triton X - 100。
