T-state hemoglobin with four ligands bound.

Flash photolysis kinetics have been measured for ligand recombination to hemoglobin (Hb) in the presence of two effectors: bezafibrate (Bzf) and inositol hexakisphosphate (IHP). The combined influence of the two independent effectors leads to predominantly T-state behavior. Samples equilibrated with 0.1 atm of CO are fully saturated, yet after photodissociation they show only T-state bimolecular recombination rates at all photolysis levels; this indicates that the allosteric transition from R to T occurs before CO rebinding and that the allosteric equilibrium favors the T-state tetramer with up to three ligands bound. Since all four ligands bind at the rate characteristic for the T-state, the return transition from T to R must occur after the fourth ligand was bound. At 1 atm of CO, rebinding to the initial R state competes with the allosteric transition resulting in a certain fraction of CO bound at the rate characteristic for the R state; this fraction is greater the smaller the percentage dissociation. Under 1 atm of oxygen, samples are not more than 93% saturated and show mainly T-state kinetics. The results show that all four hemes can bind oxygen or CO ligands in the T structure. The fraction of the kinetics occurring as geminate is less for partially liganded (T-state) samples than for fully liganded (R-state) Hb.