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

The molecular mechanism of hemoglobin cooperativity was studied kinetically by flash photolysis on mixed-state hemoglobins which consist of three ferrous carboxy subunits and one 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 hemoglobin conformational transition rate was observed to depend on its state of ligation. At 22 degrees C, pH 7, and 0.1 M phosphate, the deoxy R yields T conformational change rate is 4 x 10(4)s-1. The rate decreases to 1.4 x 10(4)s-1 for singly ligated hemoglobin. The R yields T conformation change alters the energy separation between high- and low-spin states for azidomet, cyanatomet, and thiocyanatomet subunits by about 700, 300, and 300 cal/mol, respectively. There are two possible implications of this result: (1) the iron atom spin state is not the only major factor in the determination of its position with respect to the heme plane or (2) the change with conformation of the protein force exerted by the proximal histidine on the iron atom (for an iron to heme-plane displacement of less than 0.3 A) is less than 50% of that expected from simple models in which this motion is responsible for cooperativity.