Abstract

¹H NMR spectra of the met-azide complexes of the 2 allosteric monomeric Hbs of the larvae of Chironomus thummi thummi were recorded, assigned, and analyzed. Both the magnitude of the heme Me shifts and their anomalous temp. dependence indicated a rapid equil. between a low-spin (S = 1/2 and a high-spin (S = 5/2) state. By use of the mean Me hyperfine shift as an indicator of the position of the spin equil., the axial ligand field was shown to be 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 were assigned for the met-azide complexes in both Chironomus Hb and sperm whale myoglobin, and their magnitude reflected a similar percent high-spin component as that derived from the mean heme Me shift. The pH dependence of the hyperfine shifts reflected a pK consistent with the Bohr effect. The change in percent high spin in the T « R transition was too small to account for the Bohr effect. The difference in the position at equil. for the 2 heme orientations, however, suggested that the 2 compds. may exhibit different amplitudes of the Bohr effect.