CO BINDING AND VALENCY EXCHANGE IN ASYMMETRIC HB HYBRIDS

There remains a major controversy concerning the properties of asymmet ric hemoglobin hybrids, that is, doubly liganded tetramers consisting of an unliganded dimer and a liganded dimer. Different experimental ev idence leads to opposing conclusions. Based on dimer-tetramer equilibr ium studies, special ''T-like'' properties were assigned to this hybri d (species 21), while the other biliganded tetramers were considered a s similar to fully liganded Hb [Ackers et al. (1992) Science 255, 54-6 3]. 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 rate s differ by over an order of magnitude for the two allosteric states, the CO kinetics are a sensitive probe of the tetramer conformation. Th e results show mainly rapid R-like kinetics for CO rebinding to the as ymmetric hybrids. The second technique employs a stopped-flow apparatu s 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 mixi ng with a buffer equilibrated under CO allows observation of CO bindin g to species 21. As for the flash measurements, the kinetics show pred ominantly 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 th e long incubation times used to study the dimer-tetramer equilibrium d o not lead to a stable asymmetric hybrid, but rather a random distribu tion of oxidized subunits due to electron transfer between the iron at oms of the subunits [Shibayama et al. (1997) Biochemistry 36, 4375-438 1]. We have repeated these experiments and confirm the valency exchang e in a mixture of Hb A and S (or C) parent forms, as evidenced by comp ensating amounts of oxidation or reduction of the Hb parents.