Probing the α1β2 Interface of Human Hemoglobin by Mutagenesis

The allosteric transition of hemoglobin involves an extensive reorganization of the α1β2 interface, in which two contact regions have been identified. This paper concerns the effect of two mutations located in the “switch” (αC3 Thr → Trp) and the “flexible joint” (βC3 Trp → Thr). We have expressed and characterized one double and two single mutants: Hb αT38W/βW37T, Hb βW37T, and Hb αT38W, whose structure has been determined by crystallography. We present data on: (i) the interface structure in the two contact regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rate constant. All the mutants are tetrameric and T-state in the deoxygenated derivative. Reassociation of deoxygenated dimers is not modified by interface mutations. DeoxyHb αT38W dimerizes 30% slower than HbA; Hb βW37T and Hb αT38W/βW37T dissociate much faster. We propose a binding site for I− at the switch region. The single mutants bind O2 cooperatively; the double one is almost non-cooperative, a feature confirmed by CO binding. The functional data, analyzed with the two-state model, indicate that these mutations reduce the value of the allosteric constant L.