Greenwood C, Barber D, Parr S R, Antonini E, Brunori M, Colosimo A
Biochem J. 1978 Jul 1;173(1):11-17. doi: 10.1042/bj1730011.
The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.
在pH 7.0和25摄氏度条件下，采用停流技术研究了抗坏血酸还原的假单胞菌细胞色素氧化酶与氧气的反应。观察到的时间进程很复杂，反应包括三个阶段。其中，只有最快的过程，二级速率常数为3.3×10⁴ M⁻¹·s⁻¹，依赖于氧气浓度。另外两个较慢的过程是一级反应，速率分别为1.0±0.4 s⁻¹和0.1±0.03 s⁻¹。动力学滴定实验表明，该酶对氧气的亲和常数相对较低，约为10⁴ M⁻¹。测定了所有三个反应阶段的动力学差光谱，显示每个阶段都有不同的特征。快速阶段的差光谱表明，在此过程中酶的血红素c和血红素d1成分都发生了变化。这些变化与血红素c被氧化一致，但血红素d1呈现出一种与正常氧化状态不符的形式，即使在反映血红素d1成分进一步变化的第二个动力学阶段之后，这种情况也没有恢复。根据动力学方案对结果进行了讨论。
