ABSTRACT
Cytochrome P450(BM3)-F87G reacts with aromatic aldehydes and hydrogen peroxide to generate covalent heme adducts in a reaction that may involve the formation of a stable isoporphyrin intermediate [Raner, G. M., Hatchell, A. J., Morton, P. E., Ballou, D. P., and Coon, M. J. (2000) J. Inorg. Biochem. 81, 153-160]. Electron paramagnetic resonance spectra for the proposed isoporphyrin intermediates generated using two different aromatic aldehydes suggest that, in each case, the heme remained coordinated to the apoenzyme via the cysteine thiolate, the metal center remained ferric low spin, and a slight distortion in the geometry of the pyrrole nitrogens occurred. Characterization of the resulting heme adducts via 1D and 2D NMR showed conclusively that the heme was modified at the gamma-meso position alone, and mass spectral analysis indicated loss of formate from the aldehyde prior to alkylation. The enzyme derivatives in which the hemes were covalently altered retained the characteristic UV/vis and EPR spectral properties of a P450, indicating that the heme was properly ligated in the active site. The modified enzymes were able to accept electrons from NADPH in the presence of lauric acid at a rate comparable to that of the unmodified forms, although oxidation of the lauric acid was not observed with either modified enzyme. Oxidation of 4-nitrophenol and 4-nitrocatechol was observed for both derivatives. However, 4-nitrocatechol oxidation was completely quenched in the presence of superoxide dismutase. The results are consistent with heme modification occurring through a peroxo-dependent pathway and also suggest that modification results in altered catalytic activity, rather than complete inactivation of the P450.
