Covalently Linked Heme in Cytochrome P4504A Fatty Acid Hydroxylases

Abstract

Three independent experimental methods, liquid chromatography, denaturing gel electrophoresis with heme staining, and mass spectrometry, establish that the CYP4A class of enzymes has a covalently bound heme group even though the heme is not cross-linked to the protein in other P450 enzymes. Covalent binding has been demonstrated for CYP4A1, -4A2, -4A3, -4A8, and -4A11 heterologously expressed in Escherichia coli. However, the covalent link is also present in CYP4A1 isolated from rat liver and is not an artifact of heterologous expression. The extent of heme covalent binding in the proteins as isolated varies and is substoichiometric. In CYP4A3, the heme is attached to the protein via an ester link to glutamic acid residue 318, which is on the I-helix, and is predicted to be within the active site. This is the first demonstration that a class of cytochrome P450 enzymes covalently binds their prosthetic heme group. Heme1 is the essential prosthetic group of many important proteins, including the hemoglobins, catalases, peroxidases, and cytochromes. In cytochrome P450 enzymes, the heme sits deep inside the protein with its iron ligated to a cysteine thiolate group and the sixth iron coordination site occupied by a water molecule in the substrate-free state (1). This coordination arrangement gives cytochrome P450 enzymes their unique spectral and catalytic properties. These enzymes are involved in many vital processes, including the biosynthesis of steroids and other lipophilic physiological effectors, the metabolism of drugs, and the degradation of xenobiotics. A unique feature of the cytochrome P450 enzymes is their ability to catalyze the hydroxylation of unactivated hydrocarbons under physiological conditions rather than the extreme conditions required for the reaction to occur under uncatalyzed conditions. Sequence homologies within the heme-binding region of the protein suggest that, despite the existence of numerous P450 forms with different substrate specificities, individual P450 enzymes share a common catalytic mechanism of oxygen activation.Work in this laboratory has focused on the P450 enzymes of family 4 (CYP4), especially the four known rat isoforms (CYP4A1, -4A2, -4A3, and -4A8) and the single characterized human isoform (CYP4A11) (2-5). The first three rat enzymes are present in liver and are induced by clofibrate, whereas all four enzymes are constitutively expressed in the rat kidney. The enzymes of the CYP4 family are distinguished by their unique ability to hydroxylate preferentially the thermodynamically disfavored terminal methyl group of medium and long chain fatty acids, prostaglandins, and other eicosanoids. Although other P450 enzymes hydroxylate fatty acids at internal positions of the hydrocarbon chain (6 -8), only the CYP4 enzymes preferentially hydroxylate the energetically disfavored terminal methyl group. Site-directed mutagenesis studies have begun to shed some light on the mechanism by which these enzymes achieve this specificity (4, 5, 9), but the detailed mechanism remains obscur...

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

“…Therefore, the ω-hydroxylation regiospecificity of CYP4A enzymes is of critical physiological importance. We previously (a) postulated that a constricted access channel controls ω-hydroxylation, (b) discovered that the heme is covalently bound to the protein in most CYP4 enzymes, (c) identified the residue involved in the covalent linkage, and (d) proposed that covalent heme binding is important for the architecture of the narrow channel [17,33,34]. It is thus somewhat unexpected that CYP52A21 from C. albicans predominantly hydroxylates fatty acids at the ω-position (Figure 4) despite the fact that the Glu residue involved in heme-covalent binding in CYP4A enzymes is replaced in CYP52A21 by an Ala and its heme is not covalently bound ( Fig.…”

“…Clearly, the CYP4 enzymes have evolved a strategy that allows them to specifically direct the reaction to the terminal (ω-) carbon of the fatty acid chain. We have postulated that this strategy involves a constricted access channel through which the fatty acid terminus must be threaded for oxidation, have proposed that covalent binding of the heme to the protein facilitates the assembly of a sufficiently rigid access channel, and have used terminally halogenated fatty acids to obtain information on the effective diameter of the CYP4A channel [15][16][17].…”

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

“…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).…”

“…An alignment of amino acid sequences from various members of the CYP4 family of fatty acid-oxygenating P450 enzymes was performed using the ClustalW sequence alignment program via the European Bioinformatics Institute (available at www.ebi.ac.uk/clustalw/). The sequence alignment shown is for residues in the I helix region of the P450 enzymes, surrounding the conserved glutamate residue shown to participate in covalent linkage of the heme group in eukaryotic CYP4 enzymes (23,24). The relevant amino acids at this position in the alignment are shown in boldface and are underlined.…”

“…It was shown that SDS-PAGE resolution of several CYP4 isoforms did not separate heme from the protein, suggesting that there was covalent ligation between the heme macrocycle and the protein (23,24). 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.…”