Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio Investigation of the requirements for efcient and selective cytochrome P450 monooxygenase catalysis across diferent reactions Matthew N. Podgorski a , Tom Coleman a , Rebecca R. Chao a , James J. De Voss b , John B. Bruning c , Stephen G. Bell a, ⁎ a Department of Chemistry, University of Adelaide, Adelaide, SA 5005, Australia b School of Chemistry and Molecular Bioscience, University of Queensland, St Lucia, Qld 4072, Australia c School of Biological Sciences, University of Adelaide, SA 5005, Australia ARTICLE INFO Keywords: Metalloenzyme C-H bond abstraction X-ray crystal structures Heme monooxygenase Enzyme catalysis Molecular docking ABSTRACT The cytochrome P450 metalloenzyme (CYP) CYP199A4 from Rhodopseudomonas palustris HaA2 catalyzes the highly efcient oxidation of para-substituted benzoic acids. Here we determined crystal structures of CYP199A4, and the binding and turnover parameters, with diferent meta-substituted benzoic acids in order to establish which criteria are important for efcient catalysis. When compared to the para isomers, the meta-substituted benzoic acids were less efciently oxidized. For example, 3-formylbenzoic acid was oxidized with lower activity than the equivalent para isomer and 3-methoxybenzoic acid did not undergo O-demethylation by CYP199A4. The structural data highlighted that the meta-substituted benzoic acids bound in the enzyme active site in a modifed position with incomplete loss of the distal water ligand of the heme moiety. However, for both sets of isomers the meta- or para-substituent pointed towards, and was in close proximity, to the heme iron. The absence of oxidation activity with 3-methoxybenzoic acid was assigned to the observation that the CeH bonds of this molecule point away from the heme iron. In contrast, in the para isomer they are in an ideal location for abstraction. These fndings were confrmed by using the bulkier 3-ethoxybenzoic acid as a substrate which removed the water ligand and reoriented the meta-substituent so that the methylene hydrogens pointed towards the heme, enabling more efcient oxidation. Overall we show relatively small changes in substrate structure and position in the active site can have a dramatic efect on the activity. 1. Introduction The heme-dependent cytochrome P450 (CYP) monooxygenase fa- mily of metalloenzymes catalyzes the oxidation of biological molecules often with high selectivity [1–3]. Moreover, they can catalyze CeH bond abstractions resulting in carbon hydroxylation at ambient tem- peratures using a reactive ferryl Compound I (Cpd I) intermediate [1,4–8]. The insertion of the oxygen atom into the CeH bond is hy- pothesized to occur via a radical rebound mechanism after the Cpd I intermediate abstracts a hydrogen atom from the alkyl substrate [5,9]. Importantly, members of this enzyme superfamily can catalyze a di- verse range of other oxidative transformations including epoxidations, heteroatom dealkylations, sulfoxidations and other more complex re- actions [10–16]. Investigations into the substrate range of the soluble bacterial P450 CYP199A4 revealed that the enzyme is predisposed to bind and rapidly oxidize para-substituted benzoic acid substrates [17–24]. The fastest reaction catalyzed by CYP199A4 is O-demethylation of 4-methox- ybenzoic acid, performed at a rate of 1220 min −1 with a coupling ef- fciency of 91% [17,19]. CYP199A4 is also able to bind and oxidize the bulkier substrate 3,4-dimethoxybenzoic acid (veratric acid). In this in- stance, demethylation occurs only at the para position, afording 4- hydroxy-3-methoxybenzoic acid; the meta-substituent is not attacked [21]. CYP199A4 does not hydroxylate the aromatic ring of any of these substrates and also fails to oxidize benzoic acid [19]. Crystal structures of substrate-bound CYP199A4 have been solved to elucidate the orientation of substrates in the binding pocket and examine the tendency of CYP199A4 to attack only the para-substituent (Fig. 1)[18,19,22]. These crystal structures revealed that the benzoate moiety, held almost perpendicular to the heme, forms both hydrophilic and hydrophobic interactions with residues in the binding pocket [22]. The substrate carboxylate hydrogen bonds to the hydroxyl groups of S95 and S244 and forms a salt bridge with the guanidinium of R92 [22]. Additionally, it interacts with a molecule of water which is in turn https://doi.org/10.1016/j.jinorgbio.2019.110913 Received 25 September 2019; Received in revised form 26 October 2019; Accepted 7 November 2019 ⁎ Corresponding author. E-mail address: stephen.bell@adelaide.edu.au (S.G. Bell). Journal of Inorganic Biochemistry 203 (2020) 110913 Available online 13 November 2019 0162-0134/ © 2019 Elsevier Inc. All rights reserved. T