Studies on Modified Hemoglobins: III. SPIN STATES OF FERRIC HEMOGLOBIN, SEMI-HEMOGLOBIN, AND ISOLATED SUBUNIT CHAINS

Abstract

Abstract The pH dependencies of the spin state equilibria of ferric hemoglobin, of isolated α and β subunits, and of a semi-hemoglobin were investigated by measurements of paramagnetic susceptibilities at 293° K and 77° K, visible absorption spectra at 293° K and 77° K, and electron paramagnetic resonance spectra (EPR) at 77° K and 4.2° K. Similar results were obtained by all three methods. Acid ferric forms of hemoglobin, α chains, and semi-hemoglobin, which were in the high spin state at 293° K, were reversibly converted to a mixture of high spin and low spin compounds upon cooling to 77° K. Acid ferric β chains were a mixture of high spin and low spin compounds at 293° K and in an essentially low spin state at 77° K. The low spin compound formed in these preparations was not detected by EPR at 77° K but showed EPR signals at g1 = 2.75, g2 = 2.25, and g3 = 1.69 at 4.2° K. On the basis of its optical and EPR characteristics, the low spin compound was assigned a structure in which the 6th coordination position of the heme iron is occupied by a nitrogenous group, probably by the distal histidyl residue. It was proposed that the conformational constraint exerted by the loss of the α-β subunit contacts in the β chains and the steric hindrance caused by frozen media might be responsible for the proposed bonding of the distal group directly to the heme iron.