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

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The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate (Ar-NH(2), where Ar = 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO(2)) 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 O(2) and two exogenous reducing equivalents. We recently demonstrated that a peroxodiiron(III/III) complex (peroxo- -AurF) formed by addition of O(2) to the diiron(II/II) enzyme ( -AurF) effects the initial oxidation of Ar-NH(2), generating a mu-(oxo)diiron(III/III) form of the enzyme (mu-oxo- -AurF) and (presumably) Ar-NHOH. Here we show that peroxo- -AurF also oxidizes Ar-NHOH. Unexpectedly, this reaction proceeds through to the Ar-NO(2) final product, a four-electron oxidation, and produces -AurF, with which O(2) can combine to regenerate peroxo- -AurF. Thus, conversion of Ar-NHOH to Ar-NO(2) requires only a single equivalent of O(2) and (starting from -AurF or peroxo- -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.