Calibration of the Channel That Determines the ω-Hydroxylation Regiospecificity of Cytochrome P4504A1

Abstract

The fatty acid -hydroxylation regiospecificity of CYP4 enzymes may result from presentation of the terminal carbon to the oxidizing species via a narrow channel that restricts access to the other carbon atoms. To test this hypothesis, the oxidation of 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids by recombinant CYP4A1 has been examined. Although all three 12-halododecanoic acids bind to CYP4A1 with similar dissociation constants, the 12-chloro and 12-bromo fatty acids are oxidized to 12-hydroxydodecanoic acid and 12-oxododecanoic acid, whereas the 12-iodo analogue is very poorly oxidized. Incubations in H 2 18 O show that the 12-hydroxydodecanoic acid oxygen derives from water, whereas that in the aldehyde derives from O 2 . The alcohol thus arises from oxidation of the halide to an oxohalonium species that is hydrolyzed by water, whereas the aldehyde arises by a conventional carbon hydroxylation-elimination mechanism. No irreversible inactivation of CYP4A1 is observed during 12-halododecanoic acid oxidation. Control experiments show that CYP2E1, which has an -1 regiospecificity, primarily oxidizes 12-halododecanoic acids to the -aldehyde rather than alcohol product. Incubation of CYP4A1 with 12,12-[ 2 H] 2 -12-chlorododecanoic acid causes a 2-3-fold increase in halogen versus carbon oxidation. The fact that the order of substrate oxidation (Br > Cl > > I) approximates the inverse of the intrinsic oxidizability of the halogen atoms is consistent with presentation of the halide terminus via a channel that accommodates the chloride and bromide but not iodide atoms, which implies an effective channel diameter greater than 3.90 Å but smaller than 4.30 Å.Fatty acids are readily oxidized by cytochrome P450 enzymes in an isoform-specific manner to hydroxy fatty acids and, in the case of unsaturated fatty acids, also to epoxides (1-3). The hydroxylation of fatty acids in general contributes to fatty acid homeostasis, but the oxidation of arachidonic acid gives rise to products that have specific functions in signaling pathways. For example, the -hydroxylated product, 20-hydroxyeicosatetraenoic acid (20-HETE), 1 is a vasoconstrictor (4, 5), whereas the -1 hydroxylation product 19-HETE and some of the arachidonic acid epoxides are vasodilators (2, 5, 6). The regiochemistry of oxidation of fatty acids, particularly arachidonic acid, is therefore of critical physiological importance.The oxidation of fatty acids at internal sites along the fatty acid hydrocarbon chain is mediated by a variety of cytochrome P450 enzymes. The -1 position on the carbon adjacent to the terminal carbon of the hydrocarbon chain is the usual primary default position for the oxidation of fatty acids by P450 enzymes not specifically evolved as fatty acid hydroxylases (7,8). Hydroxylation of the terminal () carbon does not occur, or is a very minor product, with such P450 enzymes. This specificity reflects the fact that the -1 position is intrinsically more reactive than the terminal () position because a secondary C-H bond is several K cal weake...

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

“…All three 12-halododecanoic acids underwent comparable oxidation of the halide atom, as indicated by formation of 12-hydroxydodecanoic acid (Table 3). Previous work has shown that the hydroxyl oxygen in this product derives from water, not molecular oxygen, and is introduced by hydrolysis of an initially formed oxohalonium (R-X + -O -) metabolite (17). However, the extent of ";ω-1" hydroxylation, which leads to the aldehyde 12-oxododecanoic acid, decreased in proportion to the size of the terminal halogen atom (i.e., Cl > Br ≫ I).…”

“…The syntheses of 12-chlorododecanoic acid, 12-bromododecanoic acid, and 12-iodododecanoic acid have been reported elsewhere [17]. Other chemicals were of the highest grade commercially available.…”

“…The derivatized samples were allowed to cool, vortexed, and transferred to sealed Teflon-capped glass vials for either manual or autoinjection into the GC-MS. Analyses were performed on an Agilent 6850 gas chromatograph coupled to an Agilent 5973 Network Mass Selective Detector in the electron impact mode as previously described [17].…”

“…12-Halododecanoic acids have been used to investigate the dimensions of the putative channel through which substrates are presented for oxidation in the mammalian enzyme CYP4A1 [17]. The P450-catalyzed oxidation of halogen-substituted carbons normally proceeds via hydroxylation of the carbon followed by elimination of the halide ion to give the carbonyl product.…”

“…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].…”