A generalization of the Szabo-Karplus statistical mechanical model for hemoglobin cooperativity is formulated. The model fits the available thermodynamic and spectroscopic data with assumptions that are consistent with structural results and empirical energy function calculations. It provides a mechanism of hemoglobin cooperativity that is a generalization of the proposals of Monod, Wyman, and Changeux and of Perutz. The role of nonsalt-bridge related sources of constraints on ligand affinity and the mode of salt-bridge coupling to tertiary-quaternary structural changes are examined within the framework of the model. Analysis of proton release data for a range of pH values indicates that a pH-independent part of cooperativity must be present. The pH dependence of the first and last Adair constants point to partial linkage of salt bridges to ligation in the deoxy state and to a destabilized intra-13chain salt bridge in the unliganded oxy state.Hemoglobin has long been regarded as the prototype system for the investigation of cooperativity in macromolecules. The essential aim of such studies is to determine the detailed relationship between structural changes induced by ligation and the thermodynamic and kinetic manifestation of cooperativity. The x-ray structures of various hemoglobins solved by Perutz and his collaborators (1) have demonstrated that there exist two quaternary structures (deoxy and oxy) for the tetramer and two tertiary structures for each individual chain (liganded and unliganded). Based on the structural results and other data, Perutz (2, 3) proposed a stereochemical mechanism for cooperativity, in which salt bridges (some with ionizable protons in the neutral pH range) provide the link between ligand-induced tertiary structural changes and the relative stability of the two quaternary forms. To examine the thermodynamic correlates of the Perutz mechanism, its elements were incorporated into a statistical-mechanical model (4) (referred to as SK in what follows) that utilizes a partition function describing the influence of homotropic (oxygen) and heterotropic (protons and 2,3-bisphosphoglycerate) effectors on the set of contributing structures. It was found that the model was able to provide a satisfactory description of the cooperativity in ligand binding, the alkaline Bohr effect, the effect of 2,3-bisphosphoglycerate on ligand affinity, and the influence of chemical modifications and certain mutations on these properties (4-6). Further, the values of the model parameters, which correspond to physically meaningful quantities, were in the range suggested by independent estimates, thus providing support for the basic postulates of the Perutz mechanism.The structural, spectroscopic, and thermodynamic data on hemoglobin that have become available since the model was proposed call for its reevaluation at this time. Of particular importance are the structural results concerning the nature of tertiary-quaternary coupling. It is now clear from comparisons of crystal structures of unliganded d...