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

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of peo ple worldwide who suffer from alpha -thalassemia. alpha -Thalassemia is a d isease in which there is a deletion of one or more of the four alpha -chain genes, and excess gamma and beta chains spontaneously form homotetramers. The gamma (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 (alpha (2)beta (2)) o r fetal (alpha (2)gamma (2)) Hbs, it has a lower affinity for 0.2 than HbH (beta (4)). To better understand the association and ligand binding propert ies of the gamma (4) tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferr ous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 Angstrom resolution (R = 21.1%, R-free = 24.4%), and that o f ferric azide (N-3(-)) Hb Bart's was similarly determined at 1.86 Angstrom resolution (R = 18.4%, R-free = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140 degrees, and with an unusually lo ng Fe-C bond of 2.25 Angstrom. This geometry is attributed to repulsion fro m the distal His63 at the low pH of crystallization (4.5). In contrast, azi de is bound to the oxidized heme iron in the methemoglobin crystals at an a ngle of 112 degrees, in a perfect orientation to accept a hydrogen bond fro m 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 dissoci ation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.