Nuclear magnetic resonance determination of ligand-induced conformational changes in myoglobin

Abstract

We report the results of a high-resolution proton nuclear magnetic resonance at 220 MHz of deoxymyoglobin and three diamagnetic complexes of myoglobin with oxygen, carbon monoxide and ethylisocyanide. We emphasize the resonances which are shifted from their normal positions by ring currents from aromatic rings, particularly the porphyrin ring, because these ring current shifts are very sensitive to changes of the relative co-ordinates of the observed protons and the ring. A specific resonance at −6.1 p.p.m., with intensity of about three protons, has been attributed to the protons of an aromatic residue, shifted upfield by the ring currents of the heme group. A possible origin of this resonance is Phe CD1. The observed change of this resonance of 0.2 p.p.m. upon oxygenation corresponds to a movement of a few tenths of an angstrom unit. Slightly larger shifts upon oxygenation of resonances attributed to methyl groups of aliphatic residues are interpreted in terms of movements of about 0.5 Å. A comparison of the complete spectra of Mb ‡ and MbO 2 shows that there are many small differences of 0.1 to 0.2 p.p.m. which indicate widespread small structural changes upon oxygenation. Differences in the ring-current-shifted resonances amongst various ligated forms including cyanoferrimyoglobin, indicate the existence of structural differences in the protein when the ligand is changed.