Proton nuclear magnetic resonance investigation of the nature of solution conformational equilibria of monomeric insect deoxyhemoglobins.

The proton nuclear magnetic resonance spectra of the three monomeric deoxyhemoglobins of the insect larva Chironomus thummi thummi have been recorded, assigned, and analyzed. In the two allosteric hemoglobins, the heme methyls and vinyl protons were assigned by specific deuterium labeling. The hyperfine-shifted proximal histidyl imidazole exchangeable protons for the three native and two deuteroheme-reconstituted hemoglobins were assigned by comparison of spectra in H2O and 2H2O. Both native and reconstituted allosteric hemoglobins exhibit two sets of interconvertible resonances indicative of two heme orientations differing by a 180 degrees rotation about the alpha-gamma-meso axis, as previously found for the met-cyano analogues [La Mar, G. N., Smith, K. M., Gersonde, K., Sick, H., & Overkamp, M. (1980) J. Biol. Chem. 255, 66]. The relative pH sensitivities of the heme resonance hyperfine shifts for the two allosteric hemoglobins and the apparent pK approximately 8 indicate that the t in equilibrium r allosteric transition, as modulated by the Bohr proton, is being observed. For the native hemoglobins, the t in equilibrium r conformational transition was found to be centered at the heme periphery, with the proximal histidyl imidazole environment insensitive to both pH and the rotational position of the heme, consistent with the absence of a pH influence on the ligation on-rate. For the deuteroheme-reconstituted allosteric hemoglobins, both the heme and axial imidazole environments sense the t in equilibrium r transition, and the histidine environments for the two components for each hemoglobin can be clearly distinguished, suggesting that the ligation on-rates may depend on both pH and heme orientation.