NMR investigation of the heme electronic structure in deoxymyoglobin possessing a fluorinated heme

Abstract

The heme electronic structures of deoxymyoglobins (deoxy-Mbs) reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-(trifluoromethyl)porphyrinatoiron(III) (7-PF), 13,17-bis(2-carboxylatoethyl)-3,7-difluoro-2,8,12,18-tetramethylporphyrinatoiron(III) (3,7-DF), and 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethylporphyrinatoiron(III) (2-MF) have been characterized by ¹H and ¹⁹F NMR. The analysis of heme methyl proton shift patterns of the hemes in their bis-cyano forms demonstrated that, owing to the substitution of a strongly electron-withdrawing perfluoromethyl group, CF₃, to porphyrin, the porphyrin π-system of 7-PF is more significantly distorted from four-fold symmetry than those of the ring-fluorinated hemes, 3,7-DF and 2-MF. The presence of the heme orientation disorder resulted in the observation of the two well-resolved ¹⁹F signals in the spectra of deoxy-Mbs possessing 7-PF and 2-MF. The ¹⁹F signals of deoxy-Mb possessing 7-PF exhibited a relatively large difference in paramagnetic shift (~30 ppm), despite their small paramagnetic shifts (~30 ppm), supporting the significant contribution of a π spin delocalization mechanism in this Mb due to the d-electron configuration derived from the ⁵E ground state. On the other hand, ¹⁹F signals of deoxy-Mbs with 3,7-DF as well as 2-MF exhibited large paramagnetic shifts (~250 ppm) with a relatively small difference in the paramagnetic shift (~20 ppm), indicating the predominant contribution of spin delocalization, due to a d-electron configuration derived from the ⁵B₂ ground state. These results demonstrate for the first time that the relative contributions of the orbital ground states derived from ⁵E and ⁵B₂ states to the heme electronic structure in deoxy-Mb are affected by the distortion of the porphyrin π-system exerted by chemical properties of the heme peripheral side-chains.