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Abstract

Abstract 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.