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

用红外光谱和共振拉曼光谱研究了含氧酶和含氧酶的羰基络合物的电子结构。通过细胞色素P450&lt；CaM&gt；与其他酶的结果比较，得出P450&lt；CaM&gt；的氧活化机理如下：1)底物的效应作用；通过分析底物对P450&lt；CaM&gt；-CO络合物Fe-CO伸长的影响，发现底物、樟脑与Fe-CO部分相互作用，从而削弱C-O键的强度；2)P450&lt；CaM&Gt；-O_2络合物的O-O伸长；在1141dm^-1&gt；处检测到的P450_&lt；CaM&gt；-O_2复合体的O-O伸缩频率与氧化型肌红蛋白的O-O伸展频率几乎相同，表明这两种蛋白质的电子结构都是Fe^&lt；3&O_2^-。3)通过对P450_&lt；CaM&gt；-CO复合体中C-O伸展的分析，发现P450_&lt；CaM&Gt；-CO与P450_&lt；CaM&Gt；-复合体的C-O伸展作用是化学计量的，但C-O键强度减弱。C^&lt；15&gt；N^-铁基P450_&lt；CaM&gt；络合物中C-N键的弱化也被核磁共振分析所证实。根据这些发现，我们推测Putidaredosin的结合通过削弱O-O键的强度促进了O-O键的断裂，这是细胞色素P450&lt；CaM&gt；氧活化氧活化的最重要的步骤。4)催化活性中间体的电子结构被认为是与过氧化物酶中间体，化合物II的电子结构相同。然而，即使对于过氧化物酶中间体，它的结构仍然不清楚。从这一角度出发，用共振拉曼光谱分析了天然、血红素和金属取代的过氧化物酶的配位结构。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,