Moody A J, Cooper C E, Rich P R
Glynn Research Institute, Bodmin, U.K.
Biochim Biophys Acta. 1991 Aug 23;1059(2):189-207. doi: 10.1016/s0005-2728(05)80204-x.
We have prepared cytochrome-c oxidase from bovine heart (using a modification of the method of Kuboyama et al. (1972) J. Biol. Chem. 247, 6375-6383) which binds cyanide rapidly, shows no kinetic distinction between the two haems on reduction by dithionite, has a Soret absorption maximum above 424 nm, and has a negligible 'g' = 12' EPR signal. On incubation at pH 6.5 this 'fast' oxidase reverts to the 'slow' ('resting') form characterised by slow cyanide binding, slow reduction of haem a3 by dithionite, a blue-shifted Soret maximum and a large 'g' = 12' signal. Incubation of 'fast' oxidase with formate produces a form of the enzyme with properties almost identical to those of 'slow' oxidase. The kinetics of formate binding to 'fast' oxidase are found to be biphasic, revealing the presence of at least two 'fast' subpopulations in our preparations. Evidence is presented that there is an equilibrium mixture of high-spin and low-spin forms of haem a3 in both 'fast' subpopulations at room temperature. Incubation of 'fast' oxidase with chloride or bromide at pH 6.5 produces forms of oxidase with much lower rates of cyanide binding. Our working hypothesis is that formate mimics a binuclear centre ligand which is present in the 'slow' form of cytochrome oxidase. Although we show that chloride and bromide can also be ligands of the binuclear centre, possibly onto CuB, we can rule out either of these being the ligand present in the 'slow' enzyme. We will argue that the 'fast' and 'slow' forms of oxidase are equivalent to the 'pulsed' and 'resting' forms of oxidase, respectively.
我们已经从牛心中制备了细胞色素c氧化酶（采用了Kuboyama等人（1972年，《生物化学杂志》247卷，6375 - 6383页）方法的一种改进），该酶能快速结合氰化物，在用连二亚硫酸盐还原时，两种血红素之间没有动力学差异，Soret吸收峰最大值在424纳米以上，且具有可忽略不计的“g” = 12的电子顺磁共振信号。在pH 6.5条件下孵育时，这种“快速”氧化酶会转变为“慢速”（“静止”）形式，其特征为氰化物结合缓慢、连二亚硫酸盐对血红素a3的还原缓慢、Soret最大值蓝移以及较大的“g” = 12信号。“快速”氧化酶与甲酸盐孵育会产生一种酶形式，其性质几乎与“慢速”氧化酶相同。发现甲酸盐与“快速”氧化酶结合的动力学是双相的，这表明我们的制剂中至少存在两个“快速”亚群。有证据表明，在室温下，两个“快速”亚群中的血红素a3都存在高自旋和低自旋形式的平衡混合物。在pH 6.5条件下，“快速”氧化酶与氯离子或溴离子孵育会产生氰化物结合速率低得多的氧化酶形式。我们的工作假设是，甲酸盐模拟了细胞色素氧化酶“慢速”形式中存在的双核中心配体。尽管我们表明氯离子和溴离子也可以是双核中心的配体，可能是结合到CuB上，但我们可以排除它们中的任何一个是“慢速”酶中存在的配体。我们将论证，氧化酶的“快速”和“慢速”形式分别等同于氧化酶的“脉冲”和“静止”形式。
