Direct observation of a novel perturbed oxyferrous catalytic intermediate during reduced putidaredoxin-initiated turnover of cytochrome P-450-CAM - Probing the effector role of putidaredoxin in catalysis
Direct observation of a novel perturbed oxyferrous catalytic intermediate during reduced putidaredoxin-initiated turnover of cytochrome P-450-CAM - Probing the effector role of putidaredoxin in catalysis
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DOI：
10.1074/jbc.m505426200
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发表时间：
2005-12-23
影响因子：
4.8
通讯作者：
Dawson, JH
中科院分区：
文献类型：
--
作者：
Glascock, MC;Ballou, DP;Dawson, JH
（1 R）（+）-还原剂结合的含氧亚铁细胞色素P450-CAM与1当量的连二亚硫酸盐还原的putidaredoxin（Pdx）的单次周转在3 ℃下通过双混合快速扫描停流光谱法监测瞬时中间体的出现。与过量的樟脑，三个连续的物种后，观察到生成氧化亚铁P450-CAM和反应相对于减少Pdx：扰动氧化亚铁衍生物，作为物种，这是一个混合物的高和低自旋Fe（III），和高自旋的铁结合酶。前两个步骤的速率，类似于140和类似于85 s（-1），分别归属于扰动的含氧铁中间体的形成和还原Pdx的电子转移。在化学计量底物的存在下，即使最终状态是低自旋铁P450-CAM，也可以看到具有相似速率的三相。这与反应期间底物被羟基化一致。通过向具有过量樟脑的预形成的还原P450-CAM中心点Pdx复合物中加入分子氧而引发的单一周转反应也导致具有类似速率的相。有人建议，形成扰动的氧亚铁中间体反映了改变的H-键合到近端的Cys，增加的氧亚铁状态的还原电位，并触发电子转移从减少Pdx。这种物质可能是Pdx对P450-CAM反应性（即在催化过程中）的效应作用的直接光谱特征。无底物的含氧亚铁酶也容易与还原的Pdx反应，表明无底物的P450-CAM不能从还原的Pdx接受电子并作为NADH氧化酶起作用完全是由于还原的Pdx不能传递第一个电子而不是第二个电子。
The single turnover of ( 1R)(+)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin ( Pdx) was monitored for the appearance of transient intermediates at 3 degrees C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, as species that was a mixture of high and low spin Fe( III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, similar to 140 and similar to 85 s(-1), were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM center dot Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity ( i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.