Wrigglesworth J M, Elsden J, Chapman A, Van der Water N, Grahn M F
Department of Biochemistry, King's College, University of London, U.K.
Biochim Biophys Acta. 1988 Dec 7;936(3):452-64. doi: 10.1016/0005-2728(88)90023-0.
(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 reduction is independent of dithionite concentration over a range 0.9 mM-131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a3/cyanide complex occurs at the same rate as a3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)
（1）已在有氰化物和无氰化物的情况下研究了来自牛心的氧化型细胞色素c氧化酶静止形式与连二亚硫酸盐的反应。在这两种情况下，在0.1 M磷酸盐（pH 7）中细胞色素a的还原速率为8.2×10⁴ M⁻¹·s⁻¹。在无氰化物时，亚铁细胞色素a₃以0.016 s⁻¹的速率（表观速率常数kobs）出现。高铁细胞色素a₃在被还原之前保持其418 nm的Soret最大吸收峰。在0.9 mM至131 mM的范围内，a₃的还原速率与连二亚硫酸盐浓度无关。在有氰化物存在时，可见光谱和电子顺磁共振（EPR）光谱变化表明，高铁a₃/氰化物复合物的形成速率与无氰化物时a₃的还原速率相同。g = 3.6的信号与g = 6信号的衰减同时出现。在添加连二亚硫酸盐后，无论有无氰化物，均未检测到可归因于大量铜的EPR信号。（2）在用抗坏血酸/四甲基对苯二胺诱导周转后的不同时间向细胞色素氧化酶中添加连二亚硫酸盐，导致细胞色素a₃的双相还原，周转时间越长，快速还原相的比例增加。同时，高铁细胞色素a₃的主要稳态物种从高自旋转变为低自旋，细胞色素a的稳态还原水平下降，表明酶分子群体向周转快速的物种转变。在最终的活化形式中，快速的内部电子传递至细胞色素a₃不需要氧气。此外，在有氰化物存在的厌氧条件下，氧气不会在静止的细胞色素氧化酶还原样品中诱导进一步的电子摄取。这两个发现均与某些质子转运的O环类型机制的预测相反。（3）在厌氧条件下，使用四甲基对苯二胺或细胞色素c测量有氰化物存在时电子进入细胞色素氧化酶静止形式的情况，结果表明每个氧化酶有三个电子在约+200 mV的氧化还原电位以下进入。最初快速进入两个电子，随后第三个电子缓慢进入（表观速率常数kobs约为0.02 s）。
