Abstract
We have studied the hydrogen-exchange kinetics of hemoglobin A0 as a function of ligand, CO, saturation. In the noncooperative system, azide binding to methemoglobin, the alterations in exchange kinetics are proportional to the average degree of ligation. However, in the case of CO binding to deoxyhemoglobin the changes in hydrogen-exchange pattern run ahead of the degree of ligation. The data can be best fitted assuming that all the liganded species, regardless of the number of ligands, show the same exchange properties. This two-state behavior must be the consequence of the fact that all the conformational changes leading to increased solvent accessibility take place when the first ligand is bound. Studies of the effect of pH changes and carbamoylation on the relationship between ligand binding and hydrogen exchange show that the observed differences of hydrogen exchange between deoxy and the liganded state are linked to the alkaline Bohr effect and to the state of the alpha-N-termini. As a consequence, at pH 9 despite a highly cooperative ligand binding isotherm the differences in hydrogen exchange between the deoxy and fully liganded species have vanished. We have to conclude that the hydrogen exchange is mirroring only the first part of the overall R to T transition. In all the experiments with pH changes and carbamoylation it is the liganded form that shows changes becoming more like the deoxy state. This is not consistent with a model where ligand binding removes a structural restriction in the less accessible deoxy state.
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