Regioselective Peroxo-Dependent Heme Alkylation in P450_BM3-F87G by Aromatic Aldehydes:  Effects of Alkylation on Catalysis

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.

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors☆

“…Recent studies by Jung et al, involving freeze-quench EPR analysis of intermediates of cytochrome P450 BM3 generated using peracetic acid, suggested the formation of protein radical intermediates similar to those observed in P450 CAM ; however, no Cpd I was observed in these studies [14]. A mutant form of P450 BM3 , BM3-F87G, has recently been shown to favorably bind certain aromatic substrates, and was used in a previous stopped-flow investigation of the reaction of P450 with aromatic aldehydes and hydrogen peroxide [18,19]. Those studies showed that adducts to the heme resulted from the reactions.…”

Defluorination of 4-fluorophenol by cytochrome P450BM3-F87G: activation by long chain fatty aldehydes

“…The reactions were quenched with the addition of 400 μl 3 % (w/v) perchloric acid and placed on ice for 10 min. Samples were centrifuged to remove precipitated protein and 500 μl of the supernatant for each reaction was analysed by HPLC as described previously (Raner et al 2002). Samples were injected in an initial mobile phase consisting of H2O/acetonitrile/trifluoroacetic acid (94.9:5:0.1, by vol) at 1.5 ml min−1.…”

“…The assays for p-nitrophenoxydodecanoic acid de-alkylase and p-nitrophenol hydroxylase have been described in detail elsewhere (Schwaneberg et al 1999;Raner et al 2002). In brief, reactions with p-nitrophenoxydodecanoic acid were carried out at pH 8.2 in the presence of 45 μM substrate and 1 mM NADPH in a 1 ml cuvette at 25 °C.…”

“…Oxidation of p-nitrophenol to 4-nitrocatechol was carried out in 100 mM potassium phosphate buffer (pH 7.0) using 6 mM p-nitrophenol, 1 mM NADPH and varying concentrations of 2-decenal (50-200 μM) in 200 μl. Reactions were quenched and analyzed by HPLC using the published method for 4-nitrocatechol analysis (Raner et al 2002). The conversion of benzoquinone to hydroquine by BM3-F87G was demonstrated by incubating 50 mM benzoquinone with 0.50 μM F87G in 100 μM phosphate buffer, pH 7.4 and 1 mM NADPH for 5-30 s at 37 °C.…”

Isotopic labeling of the heme cofactor in cytochrome p450 and other heme proteins

“…Purifications of P450 BM3 , P450 BM3 -F87G and DHP were carried out according to published protocols (Raner et al 2002 andOsborne et al 2006). For the two mammalian P450s, 2B4(Δ2-27) and 2E1(Δ3-29), and the NADPH P450 reductase, enzymes were not purified to homogeneity, rather crude E. coli membrane samples were prepared via cell lysis, centrifugation to remove the cytosol, and re-suspension of 0.5 g cell membranes in 2 ml 100 mM phosphate buffer (pH 7.4) containing 0.1 mM EDTA and 15% (v/v) glycerol.…”