Covalent Heme Binding to CYP4B1 via Glu310 and a Carbocation Porphyrin Intermediate

Abstract

Recently we found that CYP4B1, and several other members of the CYP4 family of enzymes, are covalently linked to their prosthetic heme group through an ester linkage. In the current study, we mutated a conserved CYP4 I-helix residue, E310 in rabbit CYP4B1, to glycine, alanine, and aspartate to examine the effect of these mutations on the extent of covalent heme binding and catalysis. All mutants expressed well in insect cells and were isolated as a mixture of monomeric and dimeric forms as determined by LC/ESI-MS of the intact proteins. Rates of metabolism decreased in the order E310 > A310 >> G310 > D310, with the A310 and G310 mutants exhibiting alterations in regioselectivity for omega-1 and omega-2 hydroxylation of lauric acid, respectively. In marked contrast to the wild-type E310 enzyme, the G310, A310, and D310 mutants did not bind heme covalently. Uniquely, the acid-dissociable heme obtained from the D310 mutant contained an additional 16 amu relative to heme and exhibited the same chromatographic behavior as the monohydroxyheme species released upon base treatment of the covalently linked wild-type enzyme. Expression studies with H(2)(18)O demonstrated incorporation of the heavy isotope from the media into the monohydroxyheme isolated from the D310 mutant at a molar ratio of approximately 0.8:1. These data show (i) that E310 serves as the site of covalent attachment of heme to the protein backbone of rabbit CYP4B1; (ii) this I-helix glutamate residue influences substrate orientation in the active site of CYP4B1; and (iii) the mechanism of covalent heme attachment most likely involves a carbocation species located on the porphyrin.

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

“…Of the three mutants, the E310A mutant exhibited the highest rates of total metabolite formation but with a reduction in the preference of relative to -1 hydroxylation. Nevertheless, a preference for -hydroxylation by the E310A enzyme was retained for n-heptane and n-octane but not for n-nonane and n-decane, which is consistent with an earlier report that substitution of an alanine for Glu-310 in 4B1 reduces the preference for -hydroxylation of lauric acid relative to hydroxylation of adjacent secondary C-H bonds (21). The retention of -hydroxylase activity by the E310A enzyme for the shortest chain length substrates is likely to reflect other conserved features of the active site that restrains substrates near the heme iron in the absence of the conserved glutamic acid and compensates for changes related to the loss of the covalent linkage of the heme to the enzyme.…”

“…Covalent Linkage of the Heme and Glu-310 -The heme cofactor is the catalytic center of the protein, and in P450 4B1, the 5-methyl group of the heme is linked covalently to protein by an ester bond with Glu-310 that forms auto-catalytically (21). The ester bond is clearly evident as defined by continuous electron density shown in Fig.…”

“…Expression and Purification of Rabbit P450 4B1 Glu-310 Mutants-Plasmids for expression of CYP4B1 Glu-310 mutants in E. coli were generated by PCR amplification of FastBac clones used previously to express the corresponding mutants in insects cells (21). The corresponding segment of pCW-4B1 construct #1 plasmid for expression of the full-length P450 4B1 protein in E. coli (28) was replaced with the mutated sequence from the FastBac clones.…”

“…The presence of the glutamic acid near the heme iron leads to autocatalytic formation of an ester bond between the glutamic acid and the 5-methyl group of the heme (19 -21). This covalent linkage is likely to contribute to an active site architecture that favors -hydroxylation over ( -1)-hydroxylation by these enzymes because substitutions of alanine or glycine for the glutamic acid increase the propensity of the enzymes to catalyze -1 rather than -hydroxylation (21,22). Additionally, the related human enzymes 4F8 and 4F12 (which have glycine residues at this position) as well as human 4X1 and 4Z1 with the corresponding alanine have not been shown to exhibit a preference for -hydroxylation reactions.…”

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

“…This latter peak was most pronounced in case of h-P427Δ3, and the spectra overall looked very similar to those observed for the non-purified proteins (Supplementary Figure S1). Using an optimized method with extra reduction by addition of methyl viologen established by Zheng et al (2003) to measure CO-difference spectra, nearly all of the peak at~421 nm could be removed except for a small portion (<5%), with notable exception of h-P427Δ3 that still showed a considerable peak at~421 nm (Fig. 3).…”