Analysis of proton release in oxygen binding by hemoglobin: implications for the cooperative mechanism

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Abstract
The relationship in hemoglobin between cooperativity (dependence of the Hill constant on pH) and the Bohr effect (dependence of the mean oxygen affinity on pH) can be described by a statistical thermodynamic model [Szabo, A., and Karplus, M.(1972) J. Mol. Biol. 72, 163-197; Lee, A., and Karplus, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7055-7059]. In this model, salt bridges and other interactions serve to couple tertiary and quarternary structural changes. To test and refine the model, it is applied to analysis of the pH dependence of the tetramer Adair constants corrected for statistical factors (K4/1i'', i = 1-4). Attention is focused on the proton release of the first (.DELTA.H1+ = .vdelta. log K41''/.vdelta.pH) and last (.DELTA.H4+= .vdelta. log K44''/.vdelta.pH) oxygenation steps, where K4i'' are the Adair constants corrected for statistical factors. Measurements of .DELTA.H1+ and .DELTA.H4+ under carefully controlled conditions are reported, and good agreement between the model calculation and these experimental results is obtained. The salt bridges are found to be partially coupled to the ligation state in the deoxy quaternary structure; it is shown that a Monod-Wyman-Changeux-type model, in which the salt bridges are coupled only to quaternary structural change, is inconsistent with the data for .DELTA.H1. The significance of the present analysis for an evaluation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-734, 734-739] and other models for hemoglobin cooperativity is discussed.