Spin equilibrium and quaternary structure change in hemoglobin A. Experiments on a quantitative probe of the stereochemical mechanism of hemoglobin cooperativity

Abstract

The molecular mechanism of [human] Hb cooperativity was studied kinetically by flash photolysis on mixed-state Hb which consist of 3 ferrous carboxy subunits and 1 hybrid ferric subunit including fluoromet, azidomet, cyanatomet and thiocyanatomet. The effects of conformational transitions on the hybrid subunit were detected by kinetic absorption spectroscopy after the CO was fully photodissociated from the binding sites by a large pulse of light from a tunable dye laser. The Hb conformational transition rate was observed to depend on its state of ligation. At 22.degree. C, pH 7 and 0.1 M phosphate, the deoxy R .fwdarw. T conformational change rate is 4 .times. 104 s-1. The rate decreases to 1.4 .times. 104 s-1 for singly ligated Hb. The R .fwdarw. T conformation change alters the energy separation between the high- and low-spin states for azidomet, cyanatomet and thiocyanatomet subunits by about 700, 300 and 300 cal/mol, respectively. There are 2 possible implications of this result: the Fe atom spin state was not the only major factor in the determination of its position with respect to the heme plane or the change with conformation of the protein force exerted by the proximal histidine on the Fe atom (for an Fe to heme-plane displacement of less than 0.3 .ANG.) was less than 50% of that expected from simple models in which this motion is responsible for cooperativity.