Wilson M T, Greenwood C, Brunori M, Antonini E
Biochem J. 1975 Apr;147(1):145-53. doi: 10.1042/bj1470145.
In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.
在停流实验中，将氧化型细胞色素c氧化酶与亚铁细胞色素c在一系列氧气浓度下混合，并在不存在和存在氰化物的情况下进行实验，观察到一个快速阶段，这反映了快速达到平衡的过程。在这个阶段，根据所使用的亚铁细胞色素c的浓度，细胞色素a的每个血红素基团会氧化一到两个亚铁细胞色素分子。这一现象的原因将根据一种机制进行讨论，即所有电子都通过细胞色素a进入，而细胞色素a又与第二个位点处于快速平衡状态，该位点与“可见”铜（吸收830nm光）Cud相关（贝纳特等人，1971年）。细胞色素c2+与a3+之间反应的双分子速率常数在10(6)到10(7) M(-1)-S(-1)之间；不同制备物之间观察到了一些差异。在高铁亚铁细胞色素c浓度下，细胞色素c2+与细胞色素a3+的初始反应可以与涉及“可见”铜的反应分离，并且发现化学计量比接近每还原一个细胞色素a3+分子氧化一个细胞色素c2+分子。然而，在低高铁亚铁细胞色素c浓度下，两个位点（即细胞色素a和Cud）会同时被还原，初始反应的化学计量比更接近每还原一个细胞色素a分子氧化两个细胞色素c2+分子。830nm波段的漂白滞后于或与605nm波段的形成同时发生，且不依赖于细胞色素c浓度，而稳态下的消光则依赖于细胞色素c浓度。830nm吸收物种恢复的时间进程比605nm吸收成分的漂白快得多，并且与细胞色素c2+氧化的周转阶段平行。向氧化酶制剂中添加氰化物对观察到的细胞色素a和“可见”铜还原的化学计量比或动力学没有影响，但会抑制电子传递到另外两个位点，即细胞色素a3和不可检测的铜Cuu。
