Control of oxygenation in lipoxygenase and cyclooxygenase catalysis

Abstract

Lipoxygenases (LOX) and cyclooxygenases (COX) react an achiral polyunsaturated fatty acid with oxygen to form a chiral peroxide product of high regio- and stereochemical purity. Both enzymes employ free radical chemistry reminiscent of hydrocarbon autoxidation but execute efficient control during catalysis to form a specific product over the multitude of isomers found in the nonenzymatic reaction. Exactly how both dioxygenases achieve this positional and stereo control is far from clear. We present four mechanistic models, not mutually exclusive, that could account for the specific reactions of molecular oxygen with a fatty acid in the LOX or COX active site.

Figure 1. The lipoxygenase (LOX) and cyclooxygenase (COX) proteins

(A) LOX and COX catalysis, forming fatty acid hydroperoxide and PGH₂, respectively. (B) Biochemical characteristics of LOX and COX enzymes. (C) P. homomalla 8R-LOX with N-terminal β-barrel domain (left) and catalytic domain carrying the non-heme iron (magenta) [12]. (D) COX is a homodimer. The heme group (red) indicates the peroxidase active site, and arachidonic acid (green) is bound in the oxygenase active site.

Figure 2. Hydrogen abstraction and oxygenation

(A) Antarafacial hydrogen abstraction and addition of oxygen in LOX and COX. (B) Suprafacial reaction in cytochrome P450s. (C) The relationship of R and S chirality of oxygenations in linoleic acid. (D) The twelve possible positions of oxygenation in arachidonic acid: R and S configuration products are paired on either face of the fatty acid.

Figure 3. Reactions of fatty acid peroxyl radicals

(A)β-Fragmentation. (B) cis to trans double bond isomerization. (C) Formation of a cyclic peroxide (endoperoxide). (D) Reduction of the peroxyl radical to a hydroperoxide end product.

Figure 4. Illustration of four hypotheses for oxygenation control in COX and LOX catalysis

(A) Steric shielding: the enzyme blocks all but the desired reactive center of the pentadiene radical. (B) Oxygen channeling: oxygen is directed from the outside to the desired site of reaction of the pentadiene radical. (C) Selective peroxyl radical trapping: oxygen is free to react and β-fragment at all sites of the pentadiene. A strategically placed hydrogen donor traps only the desired product by reduction to the hydroperoxide. (D) Radical localization: enzyme-induced rotation around a carbon bond traps the radical and leaves but one carbon reactive with oxygen.

Figure 5. Structures of R- and S-lipoxygenases and modeling of arachidonic acid in the 8R-LOX active site

(A) Superposition of the X-ray structures of coral 8R-LOX (blue) [12] and mammalian 15S-LOX-1 (gold) [8]. Arachidonic acid (gray, with red carboxyl group) and molecular oxygen (green) are modeled in the active site in suitable positions for 8R-LOX catalysis. (B) Close-up of the 8R-LOX active site in the same orientation. C-10 of arachidonic acid is located at the open position of the iron coordination sphere in a suitable position for hydrogen abstraction, and O₂ is depicted in the Gly-428 pocket [12].

Figure 6. Stereocontrol of the initial 11R oxygenation in COX catalysis: key amino acids and molecular dynamic computations of available space and oxygen mobility

(A) Reaction is initiated by conduction of a radical from the heme group through Tyr-385 to C-13 of arachidonic acid (red). (B) The arachidonate C11-C15 pentadienyl radical (cyan) in the two monomers of COX-1 showing the computed free space above (blue bars) and below (red bars) showing the most free space under the 11R position [76]. (C) Representative traces of O₂ mobility during a timeframe of 0 to 500 ps (blue to red) [76]).

Figure 7. Delocalized and localized radicals

(A) Planar radicals are delocalized; tertiary-butyl substituents twist the carbons out of plane, producing a localized radical. (B) Rotation of a pentadienyl radical out of plane produces a localized or allyl radical. (C) Localized radicals are pyramidal and flip between two conformations (stereoisomers). (D) Depending on the direction of rotation of the 1,2-bond the localized radical adopts a pro-S or pro-R configuration.