Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus

The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate (Ar-NH2, where Ar = 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO₂) in the biosynthesis of the antibiotic, aureothin, by Streptomyces thioluteus. It has been reported that this net six-electron oxidation proceeds in three consecutive, two-electron steps, through p-hydroxylaminobenzoate (Ar-NHOH) and p-nitrosobenzoate (Ar-NO) intermediates, with each step requiring one equivalent of O₂ and two exogenous reducing equivalents. We recently demonstrated that a peroxodiiron(III/III) complex (peroxo-Fe₂ ^III/III-AurF) formed by addition of O₂ to the diiron(II/II) enzyme (Fe₂^II/II-AurF) effects the initial oxidation of Ar-NH₂, generating a μ-(oxo)diiron (III/III) form of the enzyme (μ-oxo-Fe₂^III/III-AurF) and (presumably) Ar-NHOH. Here we show that peroxo-Fe₂^III/III-AurF also oxidizes Ar-NHOH. Unexpectedly, this reaction proceeds through to the Ar-NO ₂ final product, a four-electron oxidation, and produces Fe ₂^II/II-AurF, with which O₂ can combine to regenerate peroxo-Fe₂^III/III-AurF. Thus, conversion of Ar-NHOH to Ar-NO₂ requires only a single equivalent of O₂ and (starting from Fe₂^II/II-AurF or peroxo-Fe ₂^III/III-AurF) is fully catalytic in the absence of exogenous reducing equivalents, by contrast to the published stoichiometry. This novel type of four-electron N-oxidation is likely also to occur in the reaction sequences of nitro-installing di-iron amine oxygenases in the biosyntheses of other natural products.