Location of the allosteric site for 2,3-bisphosphoglycerate on human oxy- and deoxyhemoglobin as observed by magnetic resonance spectroscopy.

2,3-Bisphosphoglycerate, the predominant phosphorylated metaholite of the human red blood cell, binds tightly to human deoxyhemoglobin and weakly, yet significantly, to human oxyhemoglobin. To locate the binding site(s) for 2,3-bisphosphoglycerate in both oxyand deoxyhemoglobins in solution, we have measured the paramagnetic effects of hemoglobin spin-labeled with a nitroxide radical at each of the two p-93 cysteine sulfur atoms per tetramer on the 31P-nuclei of 2,3-bisphosphoglycerate and have studied the effect of CrATP, a nonlabile paramagnetic analog of MgATP, bound at the allosteric site, on the EPR spectrum of the hemoglobin-bound nitroxide spin label. An appreciable paramagnetic effect of the hemoglobin-bound spin label on the transverse and longitudinal relaxation rates of the 31P-nuclei of 2,3-bisphosphoglycerate is observed with both deoxyand oxyhemoglobins, the effect being more than an order of magnitude greater on the transverse relaxation rates. These observations are consistent with the binding of 2,3-bisphosphoglycerate to both forms of hemoglobin. From the magnitude of the paramagnetic effects on the longitudinal and transverse nuclear relaxation rates, using the correlation time of -3 x lo-’ s obtained from the ratio of the paramagnetic effects on the longitudinal and transverse relaxation rates, which is of the same order as the rotational correlation time of the hemoglobin molecule, we estimate for both forms of hemoglobin similar NMR average distances of -15 A between the unpaired electron on the nitroxide moiety of the spin label and either of the two 31P-nuclei of the hemoglobin-bound 2,3-bisphosphoglycerate molecule. Consistent with this, we obtain a -16 A distance between the Cr3+ atom of CrATP bound at the allosteric site and the spin label in oxyhemoglobin from their dipolar interaction measured by EPR. These results suggest similar spatial location of the binding site of 2,3-bisphosphoglycerate in both forms of hemoglobin in solution.