Silvestrini M C, Tordi M G, Colosimo A, Antonini E, Brunori M
Biochem J. 1982 May 1;203(2):445-51. doi: 10.1042/bj2030445.
The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.
运用快速混合技术，在pH7和pH9.1条件下研究了铜绿假单胞菌呼吸链中细胞色素c - 551与其氧化酶之间的氧化还原反应。以还原态且结合CO的氧化酶为起始，向细胞色素c - 551还原方向的电子转移是单相的，在pH9.1时，主导的双分子速率常数为1.3（±0.2）×10⁷ M⁻¹·s⁻¹，在pH7.0时为4（±1）×10⁶ M⁻¹·s⁻¹。在相反方向，即在无氧条件下将氧化态的氧化酶与还原态的细胞色素c - 551混合时，观察到较低的吸光度变化和更复杂的动力学模式。用氧化型天青蛋白代替氧化型细胞色素c - 551时，结合CO的氧化酶中c血红素的氧化也是单相的，在pH9.1时二级速率常数为2（±0.7）×10⁶ M⁻¹·s⁻¹。通过用还原型天青蛋白作为可变底物对完全氧化的酶进行动力学滴定得到的氧化酶中c血红素的氧化还原电位，在pH7.0时为288 mV，在pH9.1时为255 mV。这与从还原态氧化酶的CO衍生物开始，用氧化型天青蛋白和氧化型细胞色素c - 551进行类似滴定中观察到的非常高的亲和力形成对比。得出以下结论：（i）在体外，天青蛋白和细胞色素c - 551作为铜绿假单胞菌呼吸链中氧化酶的还原底物时效率不同；（ii）CO与氧化酶中d1血红素的结合导致c - 血红素部分的氧化还原电位大幅下降（至少80 mV）。
