Oxygen binding by a~Fe 21 ! 2 b~Ni 21 ! 2 hemoglobin crystals

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of a and b subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of a and b hemes separately. Previous studies have characterized the oxygen binding by a~Ni 21 !2b~Fe 21 !2 crystals. Here, we have determined the three-dimensional ~3D! structure and oxygen binding of a~Fe 21 !2b~Ni 21 !2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation ~p50s! are 95 6 3 and 87 6 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 6 0.06 and 0.90 6 0.03. Analysis of the binding curves, taking into account the different projections of the a hemes along the optical directions, indicates that the oxygen affinity of a1 hemes is 1.3-fold lower than a2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the b subunits of a~Ni 21 !2b~Fe 21 !2 crystals. The average oxygen affinity of the a subunits ~p50 5 91 Torr! is about 1.2-fold higher than the b subunits ~p50 5 110 Torr!. In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.