Podgorski, M.N.Keto, A.B.Coleman, T.Bruning, J.B.De Voss, J.J.Krenske, E.H.Bell, S.G.2024-03-182024-03-182023Chemistry: A European Journal, 2023; 29(50):e202301371-1-e202301371-150947-65391521-3765https://hdl.handle.net/2440/140445Version of record online: August 2, 2023The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen- and sulfur-containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4-(thiophen-2-yl)benzoic acid and 4-(thiophen-3-yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels-Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X-ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4-(thiophen-3-yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4-(Furan-2-yl)benzoic acid was oxidized to 4-(4'-hydroxybutanoyl)benzoic acid using a whole-cell system. This reaction proceeded via a g-keto-α,β-unsaturated aldehyde species which could be trapped in vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds.en© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.cytochrome P450 enzymesenzyme mechanismHeterocyclesmetalloenzymesX-ray crystallographyOxidesBenzoic AcidThiophenesCytochrome P-450 Enzyme SystemOxidation-ReductionJournal article10.1002/chem.2023013712023-11-20648390Podgorski, M.N. [0000-0003-3238-8735]Bruning, J.B. [0000-0002-6919-1824]Bell, S.G. [0000-0002-7457-9727]