Oxygen activation mechanisms in cytochrome P450-and NADPH oxidase-catalyzed reactions

为了阐明细胞色素P450（P450）和NADPH氧化酶催化反应中的氧活化机制，我们大量制备了P450 cam和猪中性粒细胞NADPH氧化酶，并研究了它们的催化特性。制备的酶包括P450 cam的定点突变体和氧化酶的多组分。P450 cam催化d-樟脑羟基化为5-exo-，NADPH氧化酶将分子氧（O_2）转化为超氧阴离子（O_2）。在这些结果中，我们认为以下是最重要的。(1)P450 cam活性中心的Thr 252是P450 cam成为单加氧酶所必需的。因此，它被认为是酶的含氧中间体的质子供体。本研究制备了O-CH_3-Thr_（252）突变体，该突变体中Thr的OH基不能解离，不能提供质子，也不能作为酸催化剂。(2)在上述和以前报道的事实的基础上，我们假设了氧活化所需的质子转移网络的新方案（见Shimada等人）。该蛋白质的分子网络由Thr 252、H_2O、Asp 251和Lys 178/Arg 186（Kimata等）组成。(3)P450 cam的化合物I在其与间氯过苯甲酸反应期间被检测到，作为细胞色素P450类型反应中Fe（IV）=O型中间体的首次成功证明（Egawa等人）。(4)我们还研究了B淋巴细胞中的NADPH氧化酶。结果表明，O_2-产生的反应中心暴露在细胞膜的细胞表面之外（Kanegasaki等）。

To elucidate oxygen activation mechanisms in cytochrome P450 (P450) -and NADPH oxidase-catalyzed reactions, we prepared both P450cam and porcine neutrophil NADPH oxidase in large quantities and examined their catalytic properties.The enzymes prepared include site-directed mutants of P450cam and multiple components of the oxidase.P450cam catalyzes the hydroxylation of d-camphor to 5-exo-hydroxycamphor.The NADPH oxidase convertss molecular oxygen (O_2) to superoxide anion (O_2). Among the results, we believe the folowings are most important.(1) Thr252 in the active center of P450cam has been shown to be essential for the enzyme to be a monooxygenase. It was therefore considered as the proton donor to the oxygenated intermediate of the enzyme. In this study we were able to prepare O-CH_3-Thr252 mutant in which the OH group of Thr was no more dissociable and hence can not suply proton nor act as an acid catalyst.(2) On the basis of above and previously reported facts, we postulated a new scheme for the proton transfer network necessary for the oxygen activation(see Shimada et al.). The net work consists of Thr252, H_2O,Asp251 and Lys178/Arg186(Kimata et al.).(3) Compound I of P450cam was detected during its reaction with m-chloroperbenzoate serving as the first successful demonstration of Fe(IV)=O type intermediate in cytochrome P450 type of reactions(Egawa et al.).(4) We studied also an NADPH oxidase in B lymphocytes. The results suggested that the reaction center for the generation of O_2- was exposed to the outside of the cell-surface in the plasma membrane(Kanegasaki et al.).