Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences.

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethyl-porphyrinatoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using (19)F NMR and the O(2) binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in alpha- and beta- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O(2) affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O(2) affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O(2) affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.

19F NMR characterization of the thermodynamics and dynamics of the acid-alkaline transition in a reconstituted sperm whale metmyoglobin.

A 19F NMR study on the acid-alkaline transition in sperm whale myoglobin reconstituted with a perfluoromethyl heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), demonstrated that the thermodynamics of the transition is predominantly controlled by the stability of acidic form.

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.