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In 1970, Perutz (1) published a detailed mechanistic interpretation of cooperative oxygen binding to hemoglobin (Hb). Two key structural features of the protein molecule were stressed. First, in deoxy-Hb, the ferrous iron atom was high spin and out of the plane of the porphyrin ring toward the proximal histidine (F8). In the low affinity or T state quaternary conformation, movement back into the plane of the heme was restricted. This restriction was postulated to be the major cause of reduced affinity in the T quaternary state (1). Over the past 30 years, various spectral and structural studies have shown that the R to T conformational transition causes ruffling and strain on the ␣ heme group that results in large changes in its visible spectrum and can result in disruption of the ␣Fe-His(F8) bond when NO is bound (2-5). Although tacitly accepted by most workers, there is less direct spectral or structural evidence for proximal strain in ␤ subunits.In the original paper, Perutz (1) also suggested that ligand binding to T state ␤ subunits was sterically restricted by the presence of the His(E7) and Val(E11) side chains almost directly over the iron atom. As a result, Perutz (1) proposed that ligands first bind to ␣ subunits. This binding then causes the switch from the low to the high affinity quaternary state in which the ␤ subunits have a much more open ␤ distal pocket and can readily bind oxygen. Perutz's proposal of an ordered addition of ligands conflicted with results from Gibson's group. Their experimental data suggested that significant kinetic and equilibrium differences between the Hb subunits occur only for ligands containing three or more non-hydrogen atoms and that the ␤ and not ␣ subunits are more sterically accessible to large ligands such as n-butyl isocyanide, even in the T state (6 -10).A myriad of experimental approaches were developed to resolve this controversy concerning functional differences between the subunits, including the following: (i) measurements of the ligand binding properties of the isolated ␣ and ␤ chains (3); (ii) discovery or development of spectral signals for ligation of the individual subunits within intact tetramers (11); (iii) selective chemical modification or mutation of key residues in the ␣ and ␤ chains (12, 13); (iv) separation of kinetic intermediates by cryogenic electrophoresis (14); (v) x-ray diffraction analysis of partially liganded Hb crystals (15); and (vi) construction of valency and metal hybrid Hbs in which one pair of subunits has an inert metal-porphyrin group and the other O 2 -and CO-reactive heme groups (16 -24). The latter approach is the most definitive with respect to assigning observed rate and equilibrium constants since only one type of subunit is capable of reacting with ligands.Despite a large number of experiments and reports over the past 30 years, there is still no universal agreement on the differential reactivities of the individual subunits. Part of the problem is that the two-state model is an oversimplification. It is clear that multi...

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