A proton NMR investigation of the influence of distal glutamine on structural and dynamic properties of elephant metmyoglobin.

The proton NMR spectra of metmyoglobin from the Asian elephant, which has the replacement of glutamine for the usual distal histidine, are reported and analyzed. In the low pH region, we detect two interconvertible forms of the met-aquo-protein whose relative stabilities are independent of pH, but depend strongly on both temperature and solvent isotope composition. As the pH is raised, both species convert to the met-hydroxy form, as found for other myoglobins. The temperature dependence of the heme methyl shifts for both acidic protein forms indicates essentially high spin character for the iron, and the mean heme methyl shifts are interpreted as indicating one form with a very slightly weaker, and the other with a significantly stronger, axial ligand field than for the unique sperm whale met-aquo-myoglobin. The thermodynamic data for the equilibrium between the two species are consistent with differences of one hydrogen bond between coordinated water and the distal glutamine. Models are proposed where one form of the protein has not only the glutamine carboxyl oxygen acting as a hydrogen-bond acceptor, but also the amine group. We conclude that a distal glutamine can act both as a stronger and as a weaker hydrogen-bond acceptor towards coordinated water than the usual distal histidine. The relative rates of conversion of the two met-aquo-myoglobin forms to MetMbOH is found to be consistent with the proposed structures for the two forms.