Spectroscopic Evidence for a High-Spin Br-Fe(IV)-Oxo Intermediate in the α-Ketoglutarate-Dependent Halogenase CytC3 from Streptomyces

The complex of the mononuclear non-heme halogenase CytC3 from Streptomyces, Fe(II), α-ketoglutarate, bromide, and the substrate l -2-aminobutyryl- S -CytC2 reacts with O 2 to form a reaction intermediate. Variable-field, freeze-quench Mössbauer spectroscopy reveals this intermediate to be a mixture of two high-spin Fe(IV) complexes in an approximate 3.7/1 ratio. Freeze-quench Fe K-edge X-ray absorption spectroscopy provides further insight into the structure of this intermediate. A short 1.62-Å interaction between the Fe and one of its ligands is attributed to the Fe(IV)-oxo group, and a 2.43-Å interaction is assigned to the Fe−Br interaction. A significantly longer Fe−Br separation (2.53 Å) is observed in the reactant complex, consistent with lower valency of the Fe in the reactant complex. This intermediate is the first example for a Br−Fe(IV)-oxo complex in a protein and provides evidence for a unifying mechanism for Fe(II) and α-ketoglutarate-dependent dioxygenases and halogenases.