Studies on electronic structures and reactivities of an oxygenated form of heme-containing enzymes by vibrational spectroscopic methods

本文用红外和共振拉曼光谱方法研究了血红素酶的氧合物和羰基复合物的电子结构。通过对细胞色素P450_2<cam>与其它酶的研究结果的比较，提出了P450_2的氧活化机理<cam>：1）底物效应作用;通过分析底物对P450_ -CO络合物的Fe-CO伸缩的影响<cam>，发现底物樟脑与Fe-CO部分相互作用，2）P450_<cam>-O_2复合物的O-O伸缩;在1141 dm ↑ [2]处测得的P450_-O_2复合物的O-O伸缩频率与氧<cam><-1>合型肌红蛋白的O-O伸缩频率几乎相同，推测两种蛋白质的电子结构均为Fe^&lt;3+&gt;-O_2^-。通过对P450_ -CO复合物中C-O键伸缩的分析，发现putidaredoxin与<cam>P450_<cam>- CO复合物按化学计量结合，但C-O键强度减弱。通过~ 1H NMR分析还观察到C^<15>N^-铁P450_<cam>2络合<15>物中C-N键的弱化。推测putidaredoxin的结合通过削弱O-O键强度而促进O-O键断裂，这是细胞色素P450_活化氧的最重要步骤<cam>。4）催化活性中间体的电子结构被认为与过氧化物酶中间体化合物I或II等电子，该中间体的形成被假设为在氧化的P450_的单电子还原<cam>过程中。然而，即使是过氧化物酶中间体，其结构仍不清楚。从这一点来看，通过共振拉曼光谱分析了天然的，血红素和金属取代的过氧化物酶的化合物II的配位结构。结果表明，化合物II的配位结构为Fe（IV）=O，而不是Fe（IV）-OH，

The electronic structure of oxygensted and carbonyl complexes of hemecontaining enzymes has been examined by infrared and resonance Raman spectroscopic methods. By comparing the results on cytochrome P450_<cam> with those on other enzymes, the oxygen activation mechanism of P450_<cam> has been beduced as follows.1) Effector action of substrate; by analyzing the effects of a substrate on the Fe-CO stretch of P450_<cam>-CO complex, the substrate, camphor was found to interact with the Fe-CO portion, thereby weakening the strength of C-O bond.2) O-O stretch of P450_<cam>-O_2 complex; the O-O stretching frequency of P450_<cam>-O_2 complex detected at 1141 dm ^<-1> was almost identical with that for the oxygenated form of myoglobin, suggesting that the electronic strucxture of both proteins was Fe^<3+>-O_2^-.3) Effector action of putidaredoxin; by analyses of the C-O stretch in P450_<cam>-CO complex, putidaredoxin was found to bind stoichiometrically to P450_<cam>- CO complex with a weakening in the strength of the C-O bond. Weakening of the C-N bond in C^<15>N^-- ferric P450_<cam> complex was also observed by ^<15> N NMR analyses. From these findings, we speculate that the binding of putidaredoxin facilitates the O-O bond cleavage by weakening the O-O bond strength, being a most important step in the oxygen activation by cytochrome P450_<cam>.4) The electronic structure of catalytically active intermediate, whose formation has been hypothesized during the one-electron reduction of oxygenated P450_<cam>, has been considered to be isoelectronic to peroxidase intermediate, compound I of II. Even for the peroxidase intermediate, however, its structure remained unclear. From this point of view, the coordination structure of compound II of native, and heme- and metalsubstituted peroxidase was analyzed by a resonance Raman spectroscopy. The results indicate definitively that the coordination structure of compound II is Fe(IV)=O,but not Fe(IV)-OH,