The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 2. Cooperative free energies of (alphaFeCObetaFe)2 and (alphaFebetaFeCO)2 T-state tetramers.

Ligand photodissociation experiments are used to measure the prephotolysis equilibria between doubly liganded R and T quaternary conformers of the symmetric Fe-Co HbCO hybrids, (alpha(FeCO)beta(Co))(2) and (alpha(Co)beta(FeCO))(2). The free energies obtained from these data are used to calculate the cooperative free energies of the (alpha(FeCO)beta(Fe))(2) and (alpha(Fe)beta(FeCO))(2) intermediate CO-ligation states of normal hemoglobin in the T conformation, quantities important to the evaluation of current models of cooperativity. The symmetry rule model, incorporating sequential cooperativity of T-state ligand binding within an alphabeta dimer in addition to the traditional two-state cooperativity of the tetramer, predicts a larger free energy penalty for disturbing both dimers in a doubly liganded T tetramer than would be expected in the two-state model as currently formulated. (Cooperative energy penalties are simply proportional to the number of tetramer-bound ligands in the traditional two-state model.) The value found here for the energies of doubly liganded T microstates in which both dimers are perturbed, 7.9 +/- 0.3 kcal/mol, is consistent with the symmetry rule model but significantly higher than that expected (5-6 kcal/mol) in the two-state model of cooperativity.