In chemical terms, the regio- and stereoselective oxidation of unactivated hydrocarbon C-H bonds to the corresponding hydroxy (C-OH) products is the most difficult reaction catalyzed by cytochrome P450 enzymes. This substrate hydroxylation reaction is mediated by the “Compound I”-like ferryl species formed during the catalytic turnover of P450 enzymes. The Fe(IV) heme iron atom in this ferryl species is paired with a radical cation delocalized over the heme porphyrin ring, so the enzyme is two oxidation equivalents higher than the resting enzyme. The formation of this oxidizing intermediate via the catalytic cycle in has been extensively reviewed and is therefore not discussed here [1
Fig. 1 The cytochrome P450 catalytic cycle with the Compound I-like ferryl species highlighted by a blue square. The heme is represented by the iron between two bars, which stand for the porphyrin framework. RH is a hydrocarbon substrate, and ROH its alcohol (more ...)
The hydroxylation of a hydrocarbon chain is initiated by abstraction of the hydrogen atom of the C-H bond by the ferryl oxygen atom, yielding a transient carbon radical coupled to an Fe(IV)-OH catalytic intermediate. Rebound collapse of a hydroxyl radical equivalent from the iron with the substrate radical produces the alcohol and returns the enzyme to the resting ferric state (). Computational and experimental results indicate that the ease of oxidation of any given C-H bond is related to its bond strength; i.e., to the energy required to homolytically break the C-H into a carbon radical and a hydrogen radical (C-H -> C.
]. If the ferryl species in the enzyme-substrate complex can react with more than one C-H bond, the reaction will preferentially occur with the weaker C-H bond unless steric constraints or other factors prevent it. This is common in cytochrome P450 enzymes, as their active sites often bind a diversity of substrates relatively loosely and, not infrequently, in multiple orientations. The combination of ligand mobility and dynamic protein malleability can allow the ferryl species access to more than one C-H bond in the substrate.
Schematic outline of the hydroxylation of a terminal methyl group. The heme is abbreviated as two solid bars, the iron oxidation state is shown, as is a radical cation on the porphyrin in the first structure.
Oxidation of the terminal methyl of a hydrocarbon chain is referred to as ω-hydroxylation because it involves the last atom of the chain. It then follows that oxidation of the carbon next to the terminal methyl can be termed an (ω-1)-hydroxylation, and oxidation of a carbon n-atoms removed from the end of the chain an (ω-n)-hydroxylation. Based on their relative bond strengths, a tertiary C-H bond should be oxidized in preference to a secondary one, and a secondary in preference to a primary (). Thus, the terminal methyl C-H bonds are inherently more difficult to oxidize than those of secondary or tertiary C-H bonds in the hydrocarbon chain. The reactivity of C-H bonds of the terminal methyl can be increased by placing it adjacent to a double bond, an aromatic ring, or an oxygen or nitrogen atom, as these functionalities lower the energy of the resulting carbon radical by delocalizing the unpaired electron. However, here we only consider the situation in which the ferryl species differentiates between two or more hydrocarbon C-H bonds that have no adjacent activating functions. Thus, the hydroxylation of a benzylic methyl and O-dealkylation of an ether () are not encompassed by the definition of ω-hydroxylation as explored in this review. The oxidation of tert
-butyl groups in which the (ω-1)-carbon does not have an oxidizable C-H bond, as in the oxidation of terfenadone by CYP2J2 (), is also excluded [5
]. In effect, this review explores the mechanisms utilized to promote ω- over (ω-1)-hydroxylation of hydrocarbon chains.
Fig. 3 Benzylic hydroxylation of tolbutamide by CYP2C9 and O-demethylation of 4-methoxyacetanilide by CYP1A2 do not conform to the definition here of ω-hydroxylation. Furthermore, the oxidations of terfenadone by CYP2J2 and farnesol by a variety of enzymes (more ...)
Given the relative reactivities of hydrocarbon C-H bonds, P450 enzymes should preferentially catalyze (ω-1)- rather than ω-hydroxylation reactions. This is indeed observed for P450 enzymes not specifically engineered to promote ω-hydroxylation. For example, CYP2B1 (ω:ω-1 ratio 3:22) [6
] and CYP2E1 (ω:ω-1 ratio 1:11) [7
] oxidize lauric acid predominantly at the ω-1 position, CYP2A6 primarily oxidizes arachidonic acid at the ω-1 position [9
], and CYP102 oxidizes fatty acids at internal chain carbons (ω-1:ω-2:ω-3 36:30:34) with no ω-hydroxylation [10
]. However, some cytochrome P450 enzymes preferentially catalyze ω-hydroxylation reactions. These ω-hydroxylases must enforce their regiospecificity through protein-substrate interactions that either hinder access of the ferryl species to C-H bonds other than those of the terminal methyl group or alter the intrinsic ferryl reactivity. As expected from the tight control of the bound ligand that ω- regiospecificity implies, ω-hydroxylation is generally observed as a major process with substrate-specific enzymes involved in defined biological pathways. This review focuses on these latter enzymes and on our understanding of how ω-hydroxylation is promoted.