Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the β-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the β-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to Cl− were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters. Kinetic measurements of the last step (Hb4X3 + X → Hb4X4) in ligand binding showed that the βVal67(E11) → Thr mutation decreases the O2 association rate constant roughly 2-fold, has no effect on the O2 dissociation constant, has no effect on the CO association rate constant, and increases the CO dissociation rate constant roughly 2-fold. The net result of these effects is a 2-fold decrease in the equilibrium constant for both O2 and CO binding to β subunits in the triliganded intermediate. Unexpectedly, the βVal67(E11) → Thr mutation caused a 3-fold increase in the rate of CO binding to β-subunits within T-state deoxyhemoglobin.
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