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--AurF) formed by addition of O2 to the diiron(II/II) enzyme (-AurF) effects the initial oxidation of Ar-NH2, generating a μ-(oxo)diiron(III/III) form of the enzyme (μ-oxo--AurF) and (presumably) Ar-NHOH. Here we show that peroxo--AurF also oxidizes Ar-NHOH. Unexpectedly, this reaction proceeds through to the Ar-NO2 final product, a four-electron oxidation, and produces -AurF, with which O2 can combine to regenerate peroxo--AurF. Thus, conversion of Ar-NHOH to Ar-NO2 requires only a single equivalent of O2 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

Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

De novo proteins provide a unique opportunity to investigate the structure-function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron c