Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C-H bonds of organic molecules [1,2]. The catalytic cycle of these enzymes requires two protons, sourced from the surrounding solvent, and two electrons (Scheme 1) [3]. The first of these electrons is used to reduce the substrate-bound heme to the ferrous form, which enables dioxygen binding. The second electron reduces the resulting ferrous-dioxygen complex to the ferric-peroxo anion. This species is protonated on the distal oxygen to yield the ferric-hydroperoxy intermediate (compound 0; Cpd 0) [3]. A second protonation on the distal oxygen triggers the breaking of the O-O bond, generating a ferryl-oxo heme radical cation, compound I (Cpd I) [3]. The abstraction of an aliphatic C-H bond is then undertaken by Cpd I [1,[4], [5], [6], [7], [8], [9]]. After abstraction of the hydrogen atom from the substrate by Cpd I a Fe(IV)-OH species, Compound II (Cpd II) and an organic radical are generated [5,9,10]. These two species can then recombine to yield the alcohol product [10]. Under optimal reaction conditions, such as those found in physiological enzyme-catalyzed reactions, these two steps can merge into a dynamically coupled process [11].

These heme enzymes catalyze a diverse range of reactions as well as the oxidation of aliphatic and aromatic C-H bonds. Other reactions catalyzed include epoxidation, sulfoxidation, heteroatom dealkylation, C-C bond cleavage and C-C bond formation reactions [[12], [13], [14]]. Other researchers have taken advantage of their heme cofactors and adapted them to develop a range of new-to-nature catalytic reactions [[15], [16], [17]]. The oxidation of halogens (R-X) by cytochrome P450 enzymes is a much rarer activity due to the very high electronegativity of the halogen atoms. For example, dodecanoic acid is hydroxylated at the ω‑carbon when oxidized by CYP4A1 and CYP52A21 enzymes [18,19]. When a hydrogen at the ω‑carbon is substituted with a halogen, the activity changes, resulting in a partition between C-H hydroxylation and halogen oxidation. Hydroxylation adjacent to the halogen proceeds with corresponding elimination of HX (X = Cl or Br) to yield an aldehyde [18,19]. In these examples, a halonium R-X+-O− species was also formed from oxidation of the halogen, followed by hydrolysis to yield the ω-hydroxylation product and HOX. Dehalogenation reactions catalyzed by cytochrome P450 enzymes occur on chloramphenicol, where the terminal CHCl2 group undergoes hydroxylation followed by elimination of HCl [20,21]. Other substrates that undergo halogen oxidation require conditions where alternate oxidation sites are sterically or electronically disfavored. For example, the I-oxidation of an aryl iodide by rat liver P450 proceeds because the substrate was designed to block other nearby oxidizable positions and to trap the halogen oxidation metabolite [22].

CYP199A4, from the Rhodopseudomonas palustris strain HaA2, catalyzes the oxidation of para-substituted benzoic acids [[23], [24], [25], [26], [27], [28]]. The catalytic activity of the CYP199A4 system is supported by a class I electron transfer system which consists of a [2Fe-2S] ferredoxin (HaPux) and a flavin adenine dinucleotide containing ferredoxin reductase (HaPuR) [27,29]. For example, it binds 4-methoxybenzoic acid with high affinity (Kd = 0.28 μM), and oxidatively demethylates this substrate to generate 4-hydroxybenzoic acid with a product formation rate in excess of 1000 nmol.(nmol-CYP)−1.min−1 [24,26]. CYP199A4 exhibits similarly high affinities for the closely related substrates 4-methylbenzoic acid and 4-ethylbenzoic acid. Both substrates are hydroxylated by CYP199A4 at the benzylic position, but the latter also undergoes efficient desaturation to generate an alkene [26,30]. We have previously determined the crystal structures of several substrate-bound forms of CYP199A4 to interrogate different reactions such as alkene formation, aromatic hydroxylation and sulfoxidation [26,27,[30], [31], [32], [33], [34], [35], [36]]. In these structures, the benzoic acid moiety interacts with a highly specific set of active site amino acids. This results in the para-substituent being held over the heme in an ideal position for oxidation.

Here we compare the binding and oxidation of halogen substituted benzoic acids with CYP199A4 (Fig. 1). We aim to assess if this P450 enzyme can oxidize halogens, and if their presence alters the outcome of P450-catalyzed oxidations.