Electron paramagnetic resonance and oxygen binding studies of α-nitrosyl hemoglobin -: A novel oxygen carrier having no-assisted allosteric functions

alpha-Nitrosyl hemoglobin, alpha(Fe-NO)(2)beta(Fe)(2), which is frequently observed upon reaction of deoxy hemoglobin with limited quantities of NO in vitro as well as in vivo, has been synthetically prepared, and its reaction with O-2 has been investigation by EPR and thermodynamic equilibrium measurements. alpha-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O-2 binding at the heme sites of its beta-subunits. Its O-2 binding is coupled to the structural/functional transition between T- (low affinity extreme) and R- thigh affinity) states. This transition is linked to the reversible cleavage of the heme Fe-proximal His bonds in the alpha(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, alpha(Fe-NO)(2)beta(Fe)(2) is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of alpha-nitrosyl hemoglobin is comparable to that of normal hemoglobin, despite the fact that the oxygenation process involves only two ligation steps. All of these structural and functional evidences have been further confirmed by examining the reactivity of the sulfhydryl group of the Cys(beta 93) toward 4,4'-dipyridyl disulfide of several alpha-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O-2-carrying capacity, alpha-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O-2 carrier with improved features that could deliver O-2 to tissues effectively even after NO is sequestered at the heme sites of the alpha-subunits. It is concluded that the NO bound to the heme sites of the alpha-subunits of hemoglobin acts as a negative allosteric effector of Hb and thus might play a role in O-2/CO2 transport in the blood under physiological conditions.