Oligomerization and ligand binding in a homotetrameric hemoglobin: Two high‐resolution crystal structures of hemoglobin Bart's (γ 4 ), a marker for α‐thalassemia

Abstract

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from α‐thalassemia. α‐Thalassemia is a disease in which there is a deletion of one or more of the four α‐chain genes, and excess γ and β chains spontaneously form homotetramers. The γ 4 homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme–heme cooperativity. Although Hb Bart's has a higher O 2 affinity than either adult (α 2 β 2 ) or fetal (α 2 γ 2 ) Hbs, it has a lower affinity for O 2 than HbH (β 4 ). To better understand the association and ligand binding properties of the γ 4 tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 Å resolution ( R = 21.1%, R free = 24.4%), and that of ferric azide (N 3 − ) Hb Bart's was similarly determined at 1.86 Å resolution ( R = 18.4%, R free = 22.0%). In the carbonmonoxy–Hb structure, the CO ligand is bound at an angle of 140°, and with an unusually long Fe‐C bond of 2.25 Å. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112°, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.