Experimental resolution of cooperative free energies for the ten ligation states of human hemoglobin.

Tetrameric human hemoglobin can assume ten molecular forms that differ in the number and configuration of ligands bound at the four heme sites. For each of these species we have determined the cooperative free energy--i.e., the deviation in free energy of ligation from that which would obtain for the same sites binding as independent alpha and beta subunits. These cooperative free energies were resolved from measurements on the dissociation into dimers of tetramers in which each subunit is either unligated (Fe2+ deoxy) or "ligated" by conversion into the cyanomet form (Fe3+ CN). The results indicate that each hemoglobin tetramer acts as a three-level molecular switch. During the course of ligation, the total cooperative free energy (6 kcal/mol over all four binding steps) is expended in two transitions that are synchronized with particular ligation steps. Whether a cooperative energy transition occurs or not depends upon how the ligation step changes both the number and configuration of ligated subunits. The hemoglobin tetramer is thus a "combinatorial switch." The finding of three distinct free energy levels for the ten ligation states suggests the existence of three major structural forms of the hemoglobin tetramer.