Reductive Cytochrome P450 Reactions and Their Potential Role in Bioremediation

Abstract

Cytochrome P450 enzymes, or P450s, are haem monooxygenases renowned for their ability to insert one atom from molecular oxygen into an exceptionally broad range of substrates while reducing the other atom to water. However, some substrates including many organohalide and nitro compounds present little or no opportunity for oxidation. Under hypoxic conditions P450s can perform reductive reactions, contributing electrons to drive reductive elimination reactions. P450s can catalyse dehalogenation and denitration of a range of environmentally persistent pollutants including halogenated hydrocarbons and nitroamine explosives. P450-mediated reductive dehalogenations were first discovered in the context of human pharmacology but have since been observed in a variety of organisms. Additionally, P450-mediated reductive denitration of synthetic explosives has been discovered in bacteria that inhabit contaminated soils. This review will examine the distribution of P450-mediated reductive dehalogenations and denitrations in nature and discuss synthetic biology approaches to developing P450-based reagents for bioremediation.

Keywords: bioremediation; cytochrome P450; dehalogenation; denitration; synthetic biology.

FIGURE 1

The conventional cytochrome P450 catalytic cycle. Key steps are labelled with grey text. The substrate is marked in a blue oval, and oxygen atoms originating from molecular oxygen are shown in orange. The conventional cycle is shown with solid arrows, and uncoupling routes with dashed arrows. The cycle begins with the enzyme in the “resting state” where a water ligand occupies the active site and the haem iron is in the ferric state. Substrate binding displaces the water molecule and is followed by a one-electron reduction of the haem iron from Fe³⁺ to Fe²⁺, creating the opportunity for molecular oxygen to bind the ferrous haem. A second one-electron reduction creates a ferric-peroxo intermediate that is quickly protonated to a ferric-hydroperoxo species. A second protonation results in the release of water and formation of the highly reactive ferryl-oxo porphyrin radical known as “compound I.” Compound I abstracts a hydrogen atom from the substrate, briefly forming a ferryl hydroxide that recombines with the highly reactive substrate radical that resulted from the proton abstraction. The net result of this reaction cascade is monooxygenation of the substrate to form the product and return the haem iron to the ferric state. The P450 catalytic cycle can uncouple (dashed arrows) after either of the one-electron reduction steps, resulting in the release of reduced oxygen as superoxide or hydrogen peroxide. The ferryl hydroxide can also be protonated to cause uncoupling and the release of water without metabolism of the substrate.

FIGURE 2

Aerobic oxidative dehalogenation and anaerobic reductive dehalogenation by P450 enzymes. (A) In human liver microsomes, halothane is metabolised to difluoro-chloroethylene by P450 2A6 in hypoxic conditions or trifluoroacetic acid by P450 2E1 in aerobic conditions (Spracklin et al., 1996). (B) P450_CAM differentially metabolises 1,1,1,2-tetrachloroethane to either dichloroethylene in anaerobic conditions or trichloroacetaldehyde in aerobic conditions (Logan et al., 1993).

FIGURE 3

Physicochemical properties such as bond polarisation affect reductive dehalogenation. P450_CAM can fully dechlorinate trichloronitromethane through reductive reactions (A), but only partially dechlorinates 1,1,1,2-tetrachloroethane to 1,1-dichloroethylene (B). 1,1,2,2-tetrachloroethane cannot be metabolised by P450_CAM in reductive conditions (C) (Logan et al., 1993).

FIGURE 4

Reductive dechlorination of carbon tetrachloride. (A) P450 2E1 catalyses a one-electron reduction of carbon tetrachloride that results in release of chloride and formation of a carbon trichloride radical. The reaction cycle may uncouple with release of the carbon trichloride radical. A second one-electron reduction without protonation has also been reported, resulting in a carbon dichloride radical. Uncoupling events with release of radicals are indicated with dashed lines. (B) P450_CAM catalyses a two-electron reduction of carbon tetrachloride to yield carbon trichloride.

FIGURE 5

Two-stage fermentation for complete degradation of pentachloroethane. Pseudomonas putida was engineered to overexpress P450_CAM and toluene dioxygenase. Anaerobic conditions were maintained during the first stage of the fermentation, enabling reductive dechlorination of pentachloroethane to 1,1,2-trichloroethylene by P450_CAM. The gas feed was then switched from argon to oxygen, facilitating oxidative metabolism of 1,1,2-trichloroethylene by toluene dioxygenase (Wackett et al., 1994). Toluene dioxygenase oxidises 1,1,2-trichloroethylene to an unstable 1,2-dihydroxytrichloroethane intermediate that decomposes into a mixture of glyoxylate and formate (Shuying and Wackett, 1992), both of which can be incorporated into P. putida central carbon metabolism.