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 α and
β subunits. Metal hybrid hemoglobins, where the noniron
metal does not bind oxygen, provide the opportunity to
determine the oxygen affinities of α and β hemes
separately. Previous studies have characterized the oxygen
binding by α(Ni2+)2
β(Fe2+)2 crystals. Here, we
have determined the three-dimensional (3D) structure
and oxygen binding of α(Fe2+)2
β(Ni2+)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 ±
3 and 87 ± 4 Torr along the b and c
crystal axes, respectively, and the corresponding Hill
coefficients are 0.96 ± 0.06 and 0.90 ± 0.03.
Analysis of the binding curves, taking into account the
different projections of the α hemes along the optical
directions, indicates that the oxygen affinity of α1
hemes is 1.3-fold lower than α2 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 β subunits of α(Ni2+)2
β(Fe2+)2 crystals. The average
oxygen affinity of the α subunits (p50 = 91 Torr) is
about 1.2-fold higher than the β subunits (p50 = 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.