The reducing component BoxA of benzoyl-coenzyme A epoxidase from Azoarcus evansii is a [4Fe–4S] protein

BoxA is the reductase component of the benzoyl-coenzyme A (CoA) oxidizing epoxidase enzyme system BoxAB. The enzyme catalyzes the key step of an hitherto unknown aerobic, CoA-dependent pathway of benzoate metabolism, which is the epoxidation of benzoyl-CoA to the non-aromatic 2,3-epoxybenzoyl-CoA. The function of BoxA is the transfer of two electrons from NADPH to the epoxidase component BoxB. We could show recently that BoxB is a diiron enzyme, whereas here we demonstrate that BoxA harbors an FAD and two [4Fe–4S] clusters per protein monomer. The characterization of BoxA was hampered by severe oxygen sensitivity; the cubane [4Fe–4S] clusters degrade already with traces of oxygen. Interestingly, the adventitiously formed [3Fe–4S] centers could be reconstituted in vitro by adding Fe(II) and sulfide to retrieve the native cubane centers. BoxA is the first example of a reductase of this type that has an FAD and two bacterial ferredoxin-type [4Fe–4S] clusters. In other cases within the catalytically versatile family of diiron enzymes, the related reductases have plant-type ferredoxin or Rieske-type [2Fe–2S] centers only. ► BoxA acts as reductase in the benzoyl-CoA oxidizing epoxidase enzyme system BoxAB. ► Homodimeric BoxA harbors an FAD and two [4Fe–4S] clusters per protein monomer. ► The [4Fe–4S] clusters degrade with traces of O2, but could be reconstituted in vitro. ► BoxAB(C) represent a new subclass of bacterial multicomponent monooxygenases (BMO). ► Compared to BMOs, BoxA is the only dimeric reductase with FAD and [4Fe–4S] centers.