Hill B C
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Biochemistry. 1996 May 14;35(19):6136-43. doi: 10.1021/bi952486f.
The reaction of CO and O2 with fully reduced cytochrome caa3 from Bacillus subtilis has been studied by rapid reaction spectrophotometry. The fully reduced caa3 complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a3-CO. This adduct is photosensitive, and its recombination rate is proportional to CO concentration with a bimolecular value of 1.2 x 10(5)M-1 s-1. When the CO compound of the reduced complex is exposed to O2, the rate of oxidation proceeds at 0.1 s-1, which is assigned as the CO off rate. These kinetic constants give an equilibrium dissociation constant for the CO complex of 0.83 microM. Photolysis of the CO adduct in the presence of O2 reveals three reaction phases over the first 3 ms and an additional phase on the second time scale. A kinetic model is proposed in which fully reduced oxidase first combines with O2 and then electron transfer commences from both cytochrome a and a3, followed rapidly by electron input from CuA and the cytochrome c domain. An equivalent kinetic model has been used to account for the reactivity of mammalian cytochrome c oxidase in its electrostatic complex with soluble cytochrome c [Hill, B. C., (1994) J. Biol. Chem. 269, 2419-2425]. However, unlike the mitochondrial complex, the reactivity of cytochrome c in the B. subtilis caa3 complex is unaffected by ionic strength. Thus the cytochrome c moiety in the B. subtilis caa3 complex seems to be fixed in a reactive orientation by its covalent association with the rest of the oxidase complex. The pathway of electron transfer from cytochrome c to O2 appears very well conserved from B. subtilis to the mammalian respiratory chain, making the B. subtilis protein a good model to probe intersite electron transfer within the cytochrome c-cytochrome oxidase complex.
通过快速反应分光光度法研究了一氧化碳（CO）和氧气（O₂）与枯草芽孢杆菌完全还原态的细胞色素caa₃的反应。完全还原态的caa₃复合物与CO反应，产生的光谱是亚铁细胞色素a₃ - CO形成的特征光谱。这种加合物对光敏感，其重组速率与CO浓度成正比，双分子值为1.2×10⁵M⁻¹ s⁻¹。当还原复合物的CO化合物暴露于O₂时，氧化速率为0.1 s⁻¹，这被指定为CO解离速率。这些动力学常数给出了CO复合物的平衡解离常数为0.83微摩尔。在O₂存在下对CO加合物进行光解，在前3毫秒内显示出三个反应阶段，在第二个时间尺度上还有一个额外的阶段。提出了一个动力学模型，其中完全还原的氧化酶首先与O₂结合，然后电子从细胞色素a和a₃同时开始转移，随后迅速从CuA和细胞色素c结构域输入电子。一个等效的动力学模型已被用于解释哺乳动物细胞色素c氧化酶在其与可溶性细胞色素c的静电复合物中的反应性[希尔，B.C.，（1994年）《生物化学杂志》269，2419 - 2425]。然而，与线粒体复合物不同，枯草芽孢杆菌caa₃复合物中细胞色素c的反应性不受离子强度的影响。因此，枯草芽孢杆菌caa₃复合物中的细胞色素c部分似乎通过其与氧化酶复合物其余部分的共价结合而固定在一个反应性取向。从枯草芽孢杆菌到哺乳动物呼吸链，电子从细胞色素c转移到O₂的途径似乎非常保守，这使得枯草芽孢杆菌蛋白成为探测细胞色素c - 细胞色素氧化酶复合物内位点间电子转移的良好模型。
