Preparation and characterization of myoglobin reconstituted with Fe(II) oxaporphyrin: The monoanionic macrocycle provides unique cyanide binding behavior for the ferrous species

Abstract

Abstract Iron-oxaporphyrin possessing two propionate side chains ( FeOP ) was synthesized as an artificial cofactor for myoglobin. The autoxidation of FeOP provides a ferric μ-oxo bridged diiron structure. The Fe II /Fe III redox potential of FeOP dimethyl ester in acetonitrile is +310 mV vs an Ag|AgCl electrode as determined by cyclic voltammetry. The value is positively shifted by 710 mV from that of the native heme cofactor, indicating that the ferrous species is stabilized in the oxaporphyrin framework. Myoglobin reconstituted with Fe II OP was prepared in the presence of dithionite and characterized by UV–vis spectroscopy, ESI-TOF MS, and size exclusion chromatography. Interestingly, autoxidation of the reconstituted protein is found to release the cofactor from the heme pocket, suggesting that the affinity of Fe III OP for the apoprotein is dramatically reduced. Furthermore, cyanide binds to Fe II OP in the heme pocket of myoglobin with a binding constant of 1.2 × 10 4 M −1 , although native deoxymyoglobin has no affinity for cyanide. These findings demonstrate that FeOP is a new type of artificial cofactor for myoglobin which provides a ferrous species with unique characteristics.