Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains
Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains
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DOI：
10.1021/bi001719m
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发表时间：
2001-02-20
期刊：
影响因子：
2.9
通讯作者：
Roberts, GCK
中科院分区：
文献类型：
--
作者：
Gutierrez, A;Lian, LY;Roberts, GCK
用快速混合停流光谱学方法研究了NADPH对人细胞色素P450还原酶及其组分结构域中FAD和FMN氧化还原中心的还原作用。隔离的FAD结构域的减少发生在三个动力学可分辨的步骤中。第一个代表了氧化态FAD和NADPh之间电荷转移物种的快速形成(>；500 S(-1))。这之后是异构化(类似于200年S(-1))到第二个电荷转移物种，其特征是在长波区有更强烈的吸收。第三步代表氢化物从NADPH转移到FAD，并伴随着FAD结构域的色氨酸荧光的变化。黄素还原是可逆的，观察到的氢化物转移速率与NADPH浓度呈复杂的依赖关系。两个电子还原的FAD结构域通过形成弱缔合电子转移络合物与孤立的FMN结构域进行电子转移反应。NADPH降低CPR的发生没有直接的光谱证据表明电荷转移物种的形成，尽管这种物种的存在是间接推断的。第一个氢化物离子的转移导致还原酶的蓝色双半喹啉物种的积累，表明一个电子快速转移到FMN结构域。二半喹啉物种随着第二氢离子的转移而衰变。在与NADPH长时间孵育后出现第三相，当体系松弛到热力学上最稳定的状态时，第三相被分配给不同氧化还原物种之间的一系列平衡反应。与分离的FAD结构域一样，还原酶中的第一次氢化物转移显示出对NADPH浓度的复杂依赖关系。在较高的NADPh浓度下，观察到的氢化物转移速度很慢(类似于20 S(-1))，这种衰减速度归因于第二个NADPh分子结合而形成的较低活性的络合物的可逆形成。动力学数据根据本期前一篇论文中对该酶及其组成区域的电位研究进行了讨论[Munro，A.，Noble，M.，Robledo，L.，Daff，S.，and Chapman，S.(2001)BioChemical 40,1956-1963]。
The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (> 500 s(-1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization (similar to 200 s(-1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (similar to 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH, The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].