1H NMR Study of the Heme Molecular Structure in Sperm Whale Met-Aquo and Met-Imidazole Myoglobins

Abstract

Abstract The resolved heme peripheral side-chain proton resonances of sperm whale met-aquo myoglobin have been connected to the corresponding resonances in met-imidazole myoglobin by the magnetization transfer through the external ligand exchange. From the signal assignments in met-imidazole myoglobin, which have been independently obtained using conventional two-dimensional NMR and one-dimensional nuclear Overhauser effect difference spectroscopies, the observed connectivities unambiguously identified the corresponding resonances of met-aquo myoglobin. The calculation of the pseudo-contact shifts for the resonances of met-aquo myoglobin from the reported zero-field splitting constant [Y. -H. Kao, and J. T. J. Lecomte, J. Am. Chem. Soc., 115, 9754—9762 (1993) and J. Am. Chem. Soc., 116, 6991 (1994)] together with the diamagnetic shifts observed for carbonmonoxy myoglobin [B. C. Mabbutt, and P. E. Wright, Biochim. Biophys. Acta, 832, 175—185 (1985)], allowed the extraction of the contact shifts from their paramagnetic shifts. The heme peripheral propionate conformation in met-aquo myoglobin has been determined from the analysis of the contact shifts for the CαH2 proton resonances. The obtained conformations agreed well with those described by the X-ray study [T. Takano, J. Mol. Biol., 110, 537—568 (1977)]. The thermal spin equilibrium in met-imidazole myoglobin has been analyzed using the mean heme methyl proton shift as an indicator for the spin state of the protein. The equilibrium populations determined from the analysis of the NMR shift data did not agree with those determined from the magnetic susceptibility measurements [T. Iizuka, and M. Kotani, Biochim. Biophys. Acta,181, 275—286 (1969)], indicating that the heme methyl proton shifts cannot be used as quantitative probe for the thermal spin equilibrium in met-imidazole myoglobin. This would be attributed to the temperature-dependent delocalization of the unpaired electron density from the heme iron to the porphyrin π system and the axial ligands due to the perturbation on the coordination structure around the iron, exerted by the steric repulsion between the bound imidazole and the imidazole side-chain of the distal His residue, as revealed by the X-ray study [C. Lionetti, M. G. Guanziroli, F. Frigerio, P. Ascenzi, and M. Bolognesi, J. Mol. Biol., 217, 409—412 (1991)].