Oxygen detoxification by dienoyl‐CoA oxidase involving flavin/disulfide cofactors

• Published in: Molecular microbiology 2020-07, Vol.114 (1), p.17-30
• Main Authors: Schmid, Georg, Scheffen, Marieke, Willistein, Max, Boll, Matthias
• Format: Article
• Language: English
• Subjects: anaerobic aromatic degradation; Archaeoglobales - enzymology; Archaeoglobales - genetics; Archaeoglobales - metabolism; Aromatic compounds; benzoyl‐CoA reductase; Catalytic Domain - physiology; Cofactors; Detoxification; Disulfides - metabolism; Electron transfer; Energy Metabolism - physiology; Enzymes; Flavin mononucleotide; Flavin-adenine dinucleotide; Flavins - metabolism; flavin‐dependent oxidase; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Hydrocarbons, Aromatic - metabolism; Hydrolysis; Intermediates; Iron sulfides; old yellow enzyme; Oxidase; Oxidation; Oxidation-Reduction; Oxygen; Oxygen - metabolism; oxygen detoxification; Prokaryotes; Reductases; Substrates; thiol switch
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.14493

Class I benzoyl‐CoA reductases (BCRs) are oxygen‐sensitive key enzymes in the degradation of monocyclic aromatic compounds in anaerobic prokaryotes. They catalyze the ATP‐dependent reductive dearomatization of their substrate to cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA (1,5‐dienoyl‐CoA). An aromatizing 1,5‐dienoyl‐CoA oxidase (DCO) activity has been proposed to protect BCRs from oxidative damage, however, the gene and its product involved have not been identified, yet. Here, we heterologously produced a DCO from the hyperthermophilic euryarchaeon Ferroglobus placidus that coupled the oxidation of two 1,5‐dienoyl‐CoA to benzoyl‐CoA to the reduction of O2 to water at 80°C. DCO showed similarities to members of the old yellow enzyme family and contained FMN, FAD and an FeS cluster as cofactors. The O2‐dependent activation of inactive, reduced DCO is assigned to a redox thiol switch at Eo′ = −3 mV. We propose a catalytic cycle in which the active site FMN/disulfide redox centers are reduced by two 1,5‐dienoyl‐CoA (reductive half‐cycle), followed by two consecutive two‐electron transfer steps to molecular oxygen via peroxy‐ and hydroxyflavin intermediates yielding water (oxidative half‐cycle). This work identified the enzyme involved in a unique oxygen detoxification process for an oxygen‐sensitive catabolic enzyme. Dearomatizing benzoyl‐CoA reductases (BCRs) are highly oxygen‐sensitive key enzymes in aromatic compound‐degrading anaerobic prokaryotes that reduce their substrate to a cyclic 1,5‐dienoyl‐CoA. We identified a water‐forming 1,5‐dienoyl‐CoA oxidase (DCO) from the hyperthermophilic archaeon Ferroglobus placidus that protects BCR from irreversible O2‐damage. DCO belongs to the old yellow enzyme family and harbors active site FMN/disulfide cofactors that accomplish 1,5‐dienoyl‐CoA dependent O2 reduction to water without releasing H2O2.