Structures of R- and T-state hemoglobin Bassett: elucidating the structural basis for the low oxygen affinity of a mutant hemoglobin

The crystal structures of R- and T-state hemoglobin (Hb) Bassett have been determined to 2.15 and 1.80 Å resolution, respectively. Physiologically, Hb Bassett (αAsp94→Ala) is characterized by a low affinity for oxygen, a reduced Bohr effect and low cooperativity, as well as being slightly unstable (compared with normal adult hemoglobin; HbA). Comparisons between the Hb Bassett structures and previously determined R- and T-state HbA structures revealed that this mutant shares similar tertiary and quaternary structures with other Hbs. However, this analysis did identify localized structural differences between R-state Hb Bassett and R-state HbA at the α1β2 (α2β1) dimer interface and at the β-cleft. Specifically, the β-FG corner has shifted closer to the α-C helix in the mutant R structure. In addition, four intersubunit hydrogen bonds found at the α1β2 interfaces of native R-state Hb structures are abolished or weakened and subsequently replaced by two new intersubunit hydrogen bonds in R-state Hb Bassett. Remarkably, the newly formed hydrogen bonds in the R-state mutant structure are also observed in T-state Hb structures. At the β-cleft, βHis46, which is known to contribute to the Bohr effect in Hb, makes a unique hydrogen-bonding interaction with βAsn139 in the R-state Hb Bassett. Unlike the R-state mutant, the T-state Hb Bassett structure does not display any significant structural changes at both the α1β2 (α2β1) dimer interface and the β-cleft. Quite significantly, the mutation has led to removal of an interdimer repulsion involving α1Asp94 and β2Asp99. The R- and T-state structures of Hb Bassett suggest a stereochemical basis for the observed functional properties of this mutant.