Mechanisms of Cytochrome P450-Catalyzed Oxidations

Abstract

Enzymes are complex biological catalysts and are critical to life. Most oxidations of chemicals are catalyzed by cytochrome P450 (P450, CYP) enzymes, which generally utilize mixed-function oxidase stoichiometry, utilizing pyridine nucleotides as electron donors: NAD(P)H + O₂ + R → NAD(P)⁺ + RO + H₂O (where R is a carbon substrate and RO is an oxidized product). The catalysis of oxidations is largely understood in the context of the heme iron-oxygen complex generally referred to as Compound I, formally FeO³⁺, whose basis was in peroxidase chemistry. Many X-ray crystal structures of P450s are now available (≥ 822 structures from ≥146 different P450s) and have helped in understanding catalytic specificity. In addition to hydroxylations, P450s catalyze more complex oxidations, including C-C bond formation and cleavage. Enzymes derived from P450s by directed evolution can even catalyze more unusual reactions, e.g. cyclopropanation. Current P450 questions under investigation include the potential role of the intermediate Compound 0 (formally Fe^III-O₂ ^-) in catalysis of some reactions, the roles of high- and low-spin forms of Compound I, the mechanism of desaturation, the roles of open and closed structures of P450s in catalysis, the extent of processivity in multi-step oxidations, and the role of the accessory protein cytochrome b ₅. More global questions include exactly how structure drives function, prediction of catalysis, and roles of multiple protein conformations.

Keywords: Compound I; Cytochrome P450; cytochrome b5; directed evolution; enzymology; kinetics; oxidation; processivity.

Scheme 1.

General catalytic cycle for P450 reactions.^, In some cases cytochrome b₅ (b₅) can donate the electron in step 4. b₅ can also donate the electron in step 2 although not as efficiently because of the thermodynamic barrier. In some P450s (some bacterial and mitochondrial P450s) steps 2 and 4 involve electron donation from ferredoxin proteins. The exact electronic distribution in the Fe-O entities between steps 4–8 is not well established in most cases.

Scheme 2.

An example of the differences between explaining multiple reaction products by (A) a common intermediate, with variations due to the protein,^, and (B)”two-state” (actually multistate) theory..

Scheme 3.

Potential desaturation mechanisms for oxygenases. (A) P450s; (B) non-heme diiron enzymes; (C) iron/α-ketoglutarate (α-KG) dioxygenases.

Scheme 4.

Processivity in multistep reactions. The enzyme (E) catalyzes the conversion of A → B → C, e.g, P450 17A1. The degree of processivity is dominated by the ratio k₄ k_-3, although other rate constants can contribute.

Scheme 5.

Some unusual reactions catalyzed by modified P450 catalysts.^,, Ts: tosyl.

Scheme 6.

A proposed mechanism of nitrene transfer for P450s.

Scheme 7.

Induced fit vs. conformational selection hypotheses to explain multiplicity of enzyme conformations involved in catalysis.