Intradimeric but not Interdimeric Interface Control the Function of Hemoglobin

Chemical modifications of the sulhydryl groups located at the α1β1 interface in tetrameric human hemoglobin -- a region usually considered inert-- altered significantly its oxygenation properties: both the affinity for oxygen and cooperative ligation were substantially reduced. Still, these derivatives responded to allosteric effectors, such as pH and organic phosphates. Electron paramagnetic resonance of the nitrosyl derivatives showed a hyperfine splitting around g ∼ 2 indicating the cleavage of the bond between the α-heme iron and the proximal histidine, which indicates the presence of a low affinity conformation of hemoglobin. Compared to native hemoglobin, a reduced dimerization was also confirmed by isothermal titration calorimetry.1H-NMR measurements, on the other hand, showed unexpected results. Both the O2-ligated and CO-ligated forms of the derivatives exhibited spectra typical of the R-conformation, i.e., the absence of the typical T-makers around 11 and 14 ppm even in the presence of strong allosteric effectors, whereas the unligated form of this derivative showed the typical T-makers (these signals originate from specific interdimeric interactions.)These 1H-NMR results stand diametrically opposed to the measured oxygenation characteristics and are entirely inconsistent with the classical T - R dichotomic view that seemed to describe satisfactorily the correlation structure-function of hemoglobin for decades. The studied derivatives represent a case in which there is a complete divorce between the 1H-NMR-determined “structure” and oxygenation characterized “function”.