Assempour M, Lim D, Hill B C
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Biochemistry. 1998 Jul 14;37(28):9991-8. doi: 10.1021/bi980331c.
The cytochrome caa3 complex from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory enzyme complexes. The key difference in the cytochrome caa3 complex lies in the addition of a domain, homologous with mitochondrial cytochrome c, that is fused to the C-terminal end of its subunit II. Measurements of steady-state and transient reduction kinetics have been carried out on the cytochrome caa3 complex. Reduction of the cyanide-bound enzyme with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) supports a sequence of electron transfer in which cytochromec is reduced initially, and this is followed by rapid internal electron transfer from cytochrome c to CuA and from CuA to cytochrome a. Steady-state kinetics with exogenous cytochrome c as the substrate demonstrates the capability of the cytochrome caa3 complex to act as a cytochrome c oxidase. The cytochrome c from B. subtilis is the most efficient cytochrome c of those tested. Steady-state kinetics with ascorbate-TMPD as the reductant, in the absence of exogenous cytochrome c, reveals a biphasic pattern even though only a single, covalent cytochrome c interaction site is present. The two-phase kinetics are characterized by a low activity phase associated with a high apparent affinity for TMPD and a high activity phase with a low affinity for TMPD. This pattern is observed over a wide range of ionic strengths and enzyme concentrations, and with both purified and membrane extract forms of cytochrome caa3. It is proposed that the biphasic steady-state kinetics of this oxidase, and other members of the cytochrome oxidase superfamily, do not result directly from different interactions with cytochrome c but are due to a change in the redox kinetics within the centers of the conventional oxidase unit itself. Our results will be related to models that account for the biphasic steady-state kinetics exhibited by cytochrome oxidase.
来自枯草芽孢杆菌的细胞色素caa3复合物是呼吸酶复合物细胞色素氧化酶超家族的成员。细胞色素caa3复合物的关键差异在于添加了一个与线粒体细胞色素c同源的结构域，该结构域融合到其亚基II的C末端。已对细胞色素caa3复合物进行了稳态和瞬态还原动力学测量。用抗坏血酸和N,N,N',N'-四甲基对苯二胺（TMPD）还原与氰化物结合的酶，支持了一系列电子转移过程，其中细胞色素c最初被还原，随后是从细胞色素c到CuA以及从CuA到细胞色素a的快速内部电子转移。以外源细胞色素c为底物的稳态动力学证明了细胞色素caa3复合物作为细胞色素c氧化酶的能力。枯草芽孢杆菌的细胞色素c是所测试的细胞色素c中效率最高的。在没有外源细胞色素c的情况下，以抗坏血酸 - TMPD作为还原剂的稳态动力学显示出双相模式，尽管仅存在一个共价细胞色素c相互作用位点。两相动力学的特征是与对TMPD的高表观亲和力相关的低活性阶段和对TMPD的低亲和力的高活性阶段。在广泛的离子强度和酶浓度范围内，以及细胞色素caa3的纯化形式和膜提取物形式中都观察到这种模式。有人提出，这种氧化酶以及细胞色素氧化酶超家族的其他成员的双相稳态动力学并非直接源于与细胞色素c的不同相互作用，而是由于传统氧化酶单元本身中心内氧化还原动力学的变化。我们的结果将与解释细胞色素氧化酶所表现出的双相稳态动力学的模型相关。
