CO binding and valency exchange in asymmetric Hb hybrids.

There remains a major controversy concerning the properties of asymmetric hemoglobin hybrids, that is, doubly liganded tetramers consisting of an unliganded dimer and a liganded dimer. Different experimental evidence leads to opposing conclusions. Based on dimer-tetramer equilibrium studies, special "T-like" properties were assigned to this hybrid (species 21), while the other biliganded tetramers were considered as similar to fully liganded Hb [Ackers et al. (1992) Science 255, 54-63]. We report here results for three types of experiment. In the first, the asymmetric hybrids are produced by photodissociating CO ligands from [dimer-CO/dimer-azido-met] hybrids. Since the CO association rates differ by over an order of magnitude for the two allosteric states, the CO kinetics are a sensitive probe of the tetramer conformation. The results show mainly rapid R-like kinetics for CO rebinding to the asymmetric hybrids. The second technique employs a stopped-flow apparatus to obtain a higher percentage and a longer equilibration time of the asymmetric hybrid. In this case, sodium dithionite is used to remove oxygen from a solution containing [dimer-oxy/dimer-azido-met] hybrids. After a fixed delay (but before loss of azide ligands), a second mixing with a buffer equilibrated under CO allows observation of CO binding to species 21. As for the flash measurements, the kinetics show predominantly rapid CO binding, typical of the liganded (R-state) tetramer. The rapid CO binding is not in agreement with the predictions of a T-like conformation for species 21. One possible explanation is that the long incubation times used to study the dimer-tetramer equilibrium do not lead to a stable asymmetric hybrid, but rather a random distribution of oxidized subunits due to electron transfer between the iron atoms of the subunits [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We have repeated these experiments and confirm the valency exchange in a mixture of Hb A and S (or C) parent forms, as evidenced by compensating amounts of oxidation or reduction of the Hb parents.