19F NMR study of protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin.

A novel C2-symmetric ring-fluorinated hemin, 13,17-bis(2-carboxyethyl)-2,8,12,18-tetramethyl-3,7-difluoroporphyrin atoiron(III), has been synthesized and was incorporated into sperm whale apomyoglobin to investigate protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin (Mb) using 19F NMR spectroscopy. NMR signals for 19F atoms introduced as substituents on the present heme in ferrous low-spin and high-spin and ferric low-spin complexes have been observed and their shifts sharply reflect not only the electronic nature of the heme iron, but also in-plane asymmetry of the heme electronic structure. The two-fold symmetric electronic structure of the ring-fluorinated hemin is clearly manifested in the 19F and 1H NMR spectra of its dicyano complex. The chemical equivalence of the two fluorine atoms of the heme is removed in the active site of myoglobin and the splitting of the two 19F NMR signals provides a quantitative probe for characterizing the rhombic perturbation of the heme electronic structure induced by the heme-protein interaction. The in-plane asymmetry of heme electronic structures in carbon-monoxy and deoxy Mbs have been analyzed for the first time on the basis of the shift difference between the two 19F NMR signals of the heme and is interpreted in terms of iron-ligand binding and/or the orbital ground state of the heme. A potential utility of 19F NMR, combined with the use of a symmetric fluorinated hemin, in characterizing the heme electronic structure of myoglobin in a variety of iron oxidation, spin, and ligation states, is presented.