Structure-specific model of hemoglobin cooperativity.

Abstract

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...

Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

“…For the pH-dependency studies, species 11,21, and 32 were studied as hybrids (AB) in mixture with the two parent tetramers, AA and BB, according to Scheme I. The parent tetramers for species 11 are species 01 and 23; for species 21 they are species 01 and 41; for species 32 they are species 23 and 41 (Fig.…”

“…Although numerous models have been proposed containing various mixtures of "concerted" (6) and "sequential" (7) processes (e.g., see refs. [8][9][10][11][12], further progress has awaited experimental resolution of the actual energetics of cooperativity for the 10 microstates of ligation. The recently resolved distributions (13)(14)(15) and related findings (16,17) provide a necessary information base for deducing the rules that connect tertiary and quaternary switching with hemoglobin cooperativity.…”

Evolution of allosteric models for hemoglobin

“…The SK model was generalized and revised later by Lee and Karplus (SKL) (21,22), motivated by two results. First, X-ray crystallography of the mutant hemoglobin Kansas showed that the salt bridges do not break in the crystal upon CO binding to the T quaternary structure, in sharp contrast to the prediction of the Perutz mechanism.…”

“…4) stretches by 0.9 Å upon oxygenation of the T quaternary structure in the crystal (15). 2 The relative thermodynamic weights for a single subunit of the 4 possible species of the T quaternary structure in the SKL model at neutral pH are (21,22): 1 þ mH/S þ Kr 2 p þ KrpmH/S, where m is the concentration of hydroxyl ions and H is the hydroxyl ion binding constant to the broken salt bridge. The second and fourth terms correspond to the weights of subunits with broken and neutralized salt bridges (inter-subunit in the case of a subunits and intra-subunit in the case of b subunits), and the first and third terms are the same as in the partition function at alkaline pH of equation (7).…”

Tetrameric Hemoglobin Expressed in Escherichia coli

“…Furthermore, CO binding to the reassembled recombinant deoxy tetramer was found to be monophasic under all conditions. Hb has long been studied as a model compound for many biochemical phenomena and continues to be the object of intense work to elucidate the molecular details of protein-protein recognition, allosteric regulation, ligand binding and dynamics, spectroscopy, energetics of cooperativity, and structure-function relationships (1)(2)(3). X-ray structures of liganded (R), unliganded (T) states, and with several intermediate species have been solved to high resolution and have provided much information on the detailed characterization of the mechanism of action by Hb (4 -7).…”