19F NMR Study of the Heme Orientation and Electronic Structure in a Myoglobin Reconstituted with a Ring-Fluorinated Heme

Abstract

Abstract 13,17-Bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethylporphyrinatoiron(III) was reconstituted with sperm-whale apomyoglobin to investigate the molecular and electronic structures of the heme-active site by 19F NMR spectroscopy. The detection of the nuclear Overhauser effect between the substituted fluorine atom for a heme side-chain and an amino acid side-chain proton in the reconstituted myoglobin unequivocally determines the heme orientation relative to the protein matrix. The orientation of this fluorinated hemin in the protein was found to differ by a 180° rotation of the heme plane about its C2-axis from that of structurally analogous mesohemin in the active site of myoglobin. Thus, the difference between the methyl group and fluorine atom as the peripheral side-chain at the 2-position of the porphyrin is a critical determinant for the thermodynamical stability of the heme orientational isomers. The effects of the heme orientation on the electronic structure of the active site of myoglobin in a variety of iron oxidation, spin, and ligation states were characterized by 19F NMR spectroscopy. The present study demonstrated that the 19F NMR signals observed for hemoprotein reconstituted with fluorinated hemin provides useful information on the relationship between the heme orientation relative to the protein and its electronic structure in all accessible oxidation/spin states of the protein, that can be hardly obtained by 1H NMR spectroscopy.