Proton Nuclear Magnetic Resonance Investigation of the Conformation-Dependent Spin Equilibrium in Azide-Ligated Monomeric Insect Hemoglobins

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The proton nuclear magnetic resonance spectra of the met-azide complexes of the two allosteric monomeric hemoglobins of the insect larva Chironomus thummi thummi have been recorded, assigned, and analyzed. Both the magnitude of the heme methyl shifts and their anomalous temperature dependence indicate a rapid equilibrium between a low-spin (S = ½) and a high-spin (S = 5/2) state. Using the mean methyl hyperfine shift as an indicator of the position of the spin equilibrium, we demonstrate that the axial ligand field is influenced by the heme orientational position in the heme cavity, by the protein conformational state for each heme orientation, and by the presence of a silent point mutation in the heme cavity. The proximal histidyl imidazole exchangeable protons are assigned for the met-azide complexes in both the Chironomus hemoglobin and sperm whale myoglobin, and their magnitude reflects a similar percent high-spin component as that derived from the mean heme methyl shift. The pH dependence of the hyperfine shifts reflects a pK consistent with the Bohr effect. The change in percent high spin in the t ⇌ r transition is found to be too small to account for the Bohr effect. The difference in the position of the equilibrium for the two heme orientations, however, suggests that the two compounds may exhibit different amplitudes of the Bohr effect. © 1983, American Chemical Society. All rights reserved.

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