Spectroscopic studies of the effect of ligand donor strength on the Fe-NO bond in intradiol dioxygenases

The geometric and electronic structure of NO bound to reduced protocatechuate 3,4-dioxygenase and its substrate (3,4-dihydroxybenzoate, PCA) complex have been examined by X-ray absorption (XAS), UV-vis absorption (Abs), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The results are compared to those previously published on model complexes described as {FeNO}⁷ systems in which an S = 5/2 ferric center is antiferromagnetically coupled to an S = 1 NO^-. XAS pre-edge analysis indicates that the Fe-NO units in Fe^IIIPCD{NO^-} and Fe^IIIPCD {PCA,NO^-} lack the greatly increased pre-edge intensity representative of most {FeNO}⁷ model sites. Furthermore, from extended X-ray absorption fine structure (EXAFS) analysis, the Fe^IIIPCD{NO^-} and Fe^IIIPCD{PCA,NO^-} active sites are shown to have an Fe-NO distance of at least 1.91 Å ≈0.2 Å greater than those found in the model complexes. The weakened Fe-NO bond is consistent with the overall lengthening of the bond lengths and the fact that VTVH MCD data show that NO^- → Fe^III CT transitions are no longer polarized along the z-axis of the zero-field splitting tensor. The weaker Fe-NO bond derives from the strong donor interaction of the endogenous phenolate and substrate catecholate ligands, which is observed from the increased intensity in the CT region relative to that of {FeNO}⁷ model complexes, and from the shift in XAS edge position to lower energy. As NO is an analogue of O₂, the effect of endogenous ligand donor strength on the Fe-NO bond has important implications with respect to O₂ activation by non-heme iron enzymes.