MICROPEROXIDASE H2O2-CATALYZED AROMATIC HYDROXYLATION PROCEEDS BY A CYTOCHROME-P-450-TYPE OXYGEN-TRANSFER REACTION-MECHANISM/

The mechanism of aromatic hydroxylation of aniline and phenol derivati ves in a H2O2-driven microperoxidase-8(MP8)-catalyzed reaction was inv estigated. It was shown that the reaction was not inhibited by the add ition of scavengers of superoxide anion or hydroxyl radicals, which de monstrates that the reaction mechanism differs from that of the aromat ic hydroxylation catalyzed by a horseradish peroxidase/dihydroxyfumara te system. Additional experiments with O-18-labelled (H2O2)-O-18 demon strated that the oxygen incorporated into aniline to give 4-aminopheno l originates from H2O2. Furthermore, it was found that the addition of ascorbic acid efficiently blocks all peroxidase-type reactions that c an be catalyzed by the MP8/H2O2 system, but does not inhibit the aroma tic hydroxylation of aniline and phenol derivatives. Together, these o bservations exclude reaction mechanisms for the aromatic hydroxylation that proceed through peroxidase-type mechanisms in which the Oxygen i ncorporated into the substrate originates from O-2 or H2O. The mechani sm instead seems to proceed by an initial attack of the high-valent ir on-ore intermediate of MP8 on the pi-electrons of the aromatic ring of the substrate leading to product formation by a cytochrome-P-450-type of sigma-O-addition or oxygen-rebound mechanism. This implies that MP 8, which has a histidyl and not a cysteinate fifth axial Iigand, is ab le to react by a cytochrome-P-450-like oxygen-transfer reaction mechan ism.