Autocatalytic Mechanism and Consequences of Covalent Heme Attachment in the Cytochrome P4504A Family

Abstract

The prosthetic heme group in the CYP4A family of cytochrome P450 enzymes is covalently attached to an I-helix glutamic acid residue. This glutamic acid is conserved in the CYP4 family but is absent in other P450 families. As shown here, the glutamic acid is linked, presumably via an ester bond, to a hydroxyl group on the heme 5-methyl group. Mutation of the glutamic acid to an alanine in CYP4A1, CYP4A3, and CYP4A11 suppresses covalent heme binding. In wild-type CYP4A3 68% of the heme is covalently bound to the heterologously expressed protein, but in the CYP4A3/E318D mutant, 47% of the heme is unchanged, 47% is present as noncovalently bound 5-hydroxymethylheme, and only 6% is covalently bound to the protein. In the CYP4A3/ E318Q mutant, the majority of the heme is unaltered, and <2% is covalently linked. The proportion of covalently bound heme in the recombinant CYP4A proteins increases with time under turnover conditions. The catalytic activity is sensitive in some, but not all, CYP4A enzymes to the extent of covalent heme binding. Mutations of Glu318 in CYP4A3 decrease the apparent k cat values for lauric acid hydroxylation. The key conclusions are that (a) covalent heme binding occurs via an ester bond to the heme 5-methyl group, (b) covalent binding of the heme is mediated by an autocatalytic process, and (c) fatty acid oxidation is sensitive in some CYP4A enzymes to the presence or absence of the heme covalent link.

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

“…To establish whether the enzymes purified from E. coli had covalently attached heme, wild-type and A264E protein samples (heme domain and full-length flavocytochrome) were resolved by SDS-PAGE and stained using 3,3Ј,5,5Ј-tetramethylbenzidine following the methods described by Thomas et al (32). In contrast to the results of Rettie and co-workers (23,26) and of Ortiz de Montellano et al (24,25) with various CYP4 isoforms, there was no discernible staining of the P450 BM3 heme with 3,3Ј,5,5Ј-tetramethylbenzidine that would indicate the presence of covalently bound heme. In view of previous results indicating turnover-dependent formation of the covalent linkage in CYP4 isoforms, the A264E heme domain and full-length flavocytochrome were preincubated both with and without excess arachidonic acid in the presence or absence of excess reductant (dithionite or NADPH).…”

“…In studies of rat liver CYP4A1, a conserved glutamate residue (Glu 318 in this isoform) was found to be the amino acid residue to which the heme had become esterified. The importance of the covalent linkage of heme to the activity of this P450 enzyme was demonstrated through mutagenesis of key residues, although the activity in other CYP4A isoforms may not be so sensitive to the extent of covalent ligation of the heme to the protein (24,25). Studies on mammalian CYP4A1, CYP4A3, and CYP4A11 confirmed that covalent heme linkage to the protein is autocatalytic and occurs because of esterification with the glutamate via the heme 5-methyl group (25).…”

“…The importance of the covalent linkage of heme to the activity of this P450 enzyme was demonstrated through mutagenesis of key residues, although the activity in other CYP4A isoforms may not be so sensitive to the extent of covalent ligation of the heme to the protein (24,25). Studies on mammalian CYP4A1, CYP4A3, and CYP4A11 confirmed that covalent heme linkage to the protein is autocatalytic and occurs because of esterification with the glutamate via the heme 5-methyl group (25). The formation of a porphyrin carbocation species was postulated as an intermediate in the reaction with the conserved glutamate that facilitates covalent heme ligation (26).…”

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

“…P450s in a variety of species (19), although this motif is less conserved in family members that catalyze other reactions, such as human P450s 4F12, 4F8, 4X1, and 4Z1 that do not have a glutamic acid residue corresponding to Glu-310 of 4B1. Two of these residues compensate for hydrogen bonds that are lost due to extension of the turn in helix I adjacent to the Glu-310.…”

Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

“…Interestingly, a single covalent link between the heme and protein is also found in most members of the CYP4A, -4B, and -4F classes of cytochrome P450 enzymes (3)(4)(5)(6)(7). In contrast to LPO, however, mutagenesis studies show that the heme-protein covalent bond is not essential for the catalytic activity of at least some of these P450 enzymes (8,6). A long known example of heme covalent binding is provided by cytochrome c, in which a cysteine residue is covalently bound to each of the two porphyrin vinyl groups.…”