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

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 (1)H and (19)F 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, CF(3), to porphyrin, the porphyrin pi-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 (19)F signals in the spectra of deoxy-Mbs possessing 7-PF and 2-MF. The (19)F signals of deoxy-Mb possessing 7-PF exhibited a relatively large difference in paramagnetic shift (approximately 30 ppm), despite their small paramagnetic shifts (approximately 30 ppm), supporting the significant contribution of a pi spin delocalization mechanism in this Mb due to the d-electron configuration derived from the (5)E ground state. On the other hand, (19)F signals of deoxy-Mbs with 3,7-DF as well as 2-MF exhibited large paramagnetic shifts (approximately 250 ppm) with a relatively small difference in the paramagnetic shift (approximately 20 ppm), indicating the predominant contribution of spin delocalization, due to a d-electron configuration derived from the (5)B(2) ground state. These results demonstrate for the first time that the relative contributions of the orbital ground states derived from (5)E and (5)B(2) states to the heme electronic structure in deoxy-Mb are affected by the distortion of the porphyrin pi-system exerted by chemical properties of the heme peripheral side-chains.