Protein-structural heterogeneity in a non-allosteric monomeric insect hemoglobin monitored by proton magnetic resonance spectroscopy.

Proton NMR has revealed two modes of structural heterogeneity in the monomeric hemoglobin I of Chironomus thummi thummi, CTT I; rotational disorder caused by a 180 degree rotation of the heme about the alpha, gamma-meso axis (primary heterogeneity), which varies for each preparation or reconstitution of this hemoglobin, and a 'silent' amino acid replacement [Thr/Ala exchange in position 98(FG4)] in the vicinity of the heme group, which is invariant under all experimental conditions. The heme rotational disorder (primary heterogeneity) can be removed by reconstitution of CTT I with the symmetrical protoheme III. The secondary splitting is not affected; the ratio of intensities of the two types of resonance remains constant. The 8-methyl and 3-methyl and one of the alpha-vinyl proton resonances for the major heme rotational component and the 5-methyl and 1-methyl and one of the alpha-vinyl proton resonances for the minor heme rotational component have been identified and assigned by reconstitution with deuterium-labeled heme. Decoupling experiments have been employed to assign vinyl beta protons in cis and trans position to the respective vinyl alpha protons. Hyperfine shifts for the heme protons exhibited no pH influence above pH 6, in accord with the lack of the alkaline Bohr effect. Below pH 6, pH effects are most strongly reflected by the 8-methyl and 5-methyl proton resonances possibly reflecting titration of the propionate groups.