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

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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,