The tetrameric hemoglobin from Urechis caupo is nearly ideal for studying ligation to the T-state. Our previous EXAFS study had shown that the Fe is displaced 0.35 Å from the mean plane of the porphyrin in the HbCO derivative. We have carried out detailed kinetic studies of oxygen and CO ligation as a function of temperature in order to characterize both the kinetics and thermodynamics of ligation in this hemoglobin. The entropy change associated with ligation essentially corresponds to simple immobilization of the ligand and is virtually the same as that we have determined for leghemoglobin, an extreme R-state-type hemoglobin. The low ligand affinities thus derive from small enthalpies of ligation, which can be correlated with the large out of plane displacement of the Fe. Only oxygen pulse measurements revealed kinetic evidence for cooperative oxygen binding, but a direct measurement of oxygen binding gave a Hill number of 1.3. An allosteric analysis gave L = 2.6 and c = 0.048 (oxygen) and c = 0.77 (CO). The higher affinity state in this weakly cooperative hemoglobin is denoted T*, and it is for this state that thermodynamic quantities have been determined. The small differences between T and T* in CO binding were nevertheless sufficient to allow us to measure by flash photolysis the rate of the T* → T conformational change in terms of an allosteric model. The half-time for this transition was calculated to be 8–14 ms at 20 °C.
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