The mechanism of aromatic hydroxylation of aniline and phenol derivatives in a H,O,-driven microperoxidase-8(MP8)-catalyzed reaction was investigated. It was shown that the reaction was not inhibited by the addition of scavengers of superoxide anion or hydroxyl radicals, which demonstrates that the reaction mechanism differs from that of the aromatic hydroxylation catalyzed by a horseradish peroxidase/ dihydroxyfumarate system.Additional experiments with lXO-labelled H,'*O, demonstrated that the oxygen incorporated into aniline to give 4-aminophenol originates from H,O,. Furthermore, it was found that the addition of ascorbic acid efficiently blocks all peroxidase-type reactions that can be catalyzed by the MPS/H,O, system, but does not inhibit the aromatic hydroxylation of aniline and phenol derivatives. Together, these observations exclude reaction mechanisms for the aromatic hydroxylation that proceed through peroxidase-type mechanisms in which the oxygen incorporated into the substrate originates from O2 or H,O. The mechanism instead seems to proceed by an initial attack of the high-valent iron-oxo intermediate of MP8 on the nelectrons of the aromatic ring of the substrate leading to product formation by a cytochrome-P-450-type of cT-0-addition or oxygen-rebound mechanism. This implies that MP8, which has a histidyl and not a cysteinate fifth axial ligand, is able to react by a cytochrome-P-450-like oxygen-transfer reaction mechanism.

The in vivo cytochrome P450-catalyzed aromatic hydroxylation of a series of fluorobenzenes was investigated with special emphasis on the importance of the fluorine NIH shift. The results obtained demonstrate a minor role for the NIH shift in the metabolism of the fluorobenzenes to phenolic metabolites in control male Wistar rats. These in vivo results could indicate that (1) the NIH shift is an inherently minor process for fluorine substituents or (2) it is a potentially significant process but the presumed epoxide that leads to formation of the NIH-shifted metabolite is lost to an alternative metabolic pathway. In contrast to the in vivo data, in vitro experiments showed a significant amount of an NIH-shifted metabolite for 1,4-difluorobenzene. This result eliminates the explanation that the NIH shift is an inherently minor process for fluorine substituents. Results of additional experiments presented in this paper show that the reduced tendency of fluorine-substituted benzenes to undergo an NIH shift in vivo can-at least in part-be ascribed to the possible existence of alternative pathways for metabolism of the epoxide, such as, for example, GSH conjugation, being more efficient for fluorinated than chlorinated arene oxides.

The mechanism of microperoxidase-8 (MP-8) mediated O-and N-dealkylation was investigated. In the absence of ascorbate (peroxidase mode), many unidentified polymeric products are formed and the extent of substrate degradation correlates (r 0.94) with the calculated substrate ionization potential, reflecting the formation of radical intermediates.In the presence of ascorbate (P450 mode) formation of polymeric products is largely prevented but, surprisingly, dealkylation is not affected. In addition, aromatic hydroxylation and oxidative dehalogenation is observed. The results exclude a radical mechanism and indicate the involvement of a (hydro)peroxo-iron heme intermediate in P450-type of heteroatom dealkylation.

The present study describes tH NMR T1 relaxation rate studies on fluoromethylanilines bound to the active sites of microsomal and purified cytoehromes P45o IAI and 2B1. From the data obtained, insights into the average orientation of the substrates with respect to the paramagnetic Fe 3÷ centre in the cytochromes P4so could be derived. Particular attention was paid to a possible extra relaxation pathway for methyl protons compared to the aromatic protons, due to the rotational motion of the CH3 around the ~r-C-CH3 bond. However, this effect appeared to be minimal and to result in at most a few percent underestimation of the actual distance of the methyl protons to the Fe 3÷ centre. Furthermore, the data obtained demonstrate that all aromatic protons are at about the same average distance from the paramagnetic centre. The results also demonstrate that the fluoromethylanilines are bound to the active sites of cytochromes P45o 1A1 and 2B1 in a similar way. A time-averaged orientation of the substrate with the Fe 3+ above the aromatic ring, with the 7r-orbitals of the aromatic ring and those of the porphyrin rings in a parallel position, providing possibilities for energetically favourable ~t-~r interaction defines the orientation which best fits the results of the present study. Possibilities for a flip-flop rotation around an axis in the plane of the aromatic ring can be included in this picture, as such rotations would still result in a similar average distance of all aromatic protons to the Fe 3÷ paramagnetie centre. The results obtained also indicate that possible differences in metabolite patterns resulting from conversion of the fluoromethylanilines by different cytochromes P450, especially P45o IAI and 2B1, are unlikely to be caused by a specific orientation of the substrate imposed by the substrate binding site of the enzyme.

In a previous study, the in vivo cytochrome P450-catalyzed regioselectivity of aromatic ring hydroxylation for a series of (poly)fluorobenzenes could be quantitatively predicted by the calculated frontier orbital density distribution in the aromatic ring [Rietjens et al. (1993) Biochemistry 32, 4801-4812]. However, the relative small fluorine, its size almost comparable to a hydrogen, is not expected to influence the regioselectivity of aromatic hydroxylation due to steric hindrance. The aim of the present study was to investigate the influence of substituents larger than a hydrogen or fluorine on the possibilities for hydroxylation at the adjacent carbon center. First, the in vivo regioselectivity of aromatic ring hydroxylation of a series of C4-substituted fluorobenzenes was investigated. The results obtained demonstrate that a chlorine and cyano C4 substituent do not hamper hydroxylation at the positions ortho to the C4 carbon atom. For 4-chloro- and 4-cyanofluorobenzene, the observed regioselectivity of aromatic hydroxylation correlated with the regioselectivity predicted on the basis of the frontier orbital density distribution. In contrast, a bromine and iodine substituent affected the hydroxylation at the adjacent ortho carbon centers, reducing it to respectively 40 and 6% of the calculated intrinsic reactivity of the carbon centers. Additional experiments were performed to investigate whether the regioselectivity of the aromatic hydroxylation of the C4-substituted fluorobenzene model compounds was influenced by changes in the cytochrome P450 enzyme pattern. Results obtained demonstrate that for these relatively small substrates the regioselectivity of their hydroxylation was not significantly influenced by several cytochrome P450 inducers. This suggests that the active sites of the cytochromes P450 catalyzing the aromatic hydroxylation do not impose a stereoselective orientation of the aromatic rings with respect to the iron-oxo porphyrin reaction center. Thus, the working hypothesis for additional experiments was that the deviations for the regioselectivity of aromatic hydroxylation observed for 4-bromo- and 4-iodofluorobenzene may be ascribed to steric hindrance by the bromine and iodine substituents hampering the attack of the cytochrome P450 iron-oxo species on the adjacent carbon centers in the benzene derivative. This working hypothesis was further tested by investigating whether useful steric correction factors could be derived from the results obtained with the series C4-substituted fluorobenzenes. These correction factors should make it possible to correct calculated relative reactivities of carbon sites for steric hindrance by substituents positioned ortho with respect to the carbon to be hydroxylated. This will make it possible to better explain and predict the regioselectivities for other chlorine-, bromine-, iodine-, and cyano-containing fluorobenzenes. The in vivo regioselectivity of aromatic ring hydroxylation of a series of five chlorine-, bromine-, iodine-, or cyano-containing fluorobenze...