Energetic components of the allosteric machinery in hemoglobin measured byhydrogen exchange

A hydrogen exchange (HX) functional labeling method was used to study allos terically active segments in human hemoglobin (Hb) at the alpha-chain N ter minus and the beta-chain C terminus. Allosterically important interactions that contact these segments were removed one or more at a time by mutation (Hbs Cowtown, Bunbury, Barcelona, Kariya), proteolysis (desArg141 alpha, de sHis146 beta), chemical modification (N-ethylsuccinimidyl-Cys93 beta), and the withdrawal of extrinsic effecters (phosphate groups, chloride). The eff ects of each modification on HX rate at the local and the remote position w ere measured in the deoxy Hb T-state and translated into change in structur al free energy at each position. The removal of individual salt links destabilizes local structure by 0.4 to 0.75 kcal/mol (pH 7.4, 0 degrees C, 0.35 M ionic strength) and often produ ces cross-subunit effects while hemoglobin remains in the T-state. In doubl y modified hemoglobins, different changes that break the same links produce identical destabilization, changes that are structurally independent show energetic additivity, acid changes that intersect show energetic overlap. F or the overall T-state to R-state transition and for some but not all modif ications within the T-state, the summed loss in stabilization free energy m easured at the two chain termini matches the total loss in allosteric free energy measured by global methods. These observations illustrate the import ance of evaluating the detailed energetics and the modes of energy transfer that define the allosteric machinery. (C) 1998 Academic Press.