Slow dynamics measured by phosphorescence lifetime reveals global conformational changes in human adult hemoglobin...

The mechanism underlying allostery in hemoglobin (Hb) is still not completely understood. Various models describing the action of allosteric effectors on Hb function have been published in the literature. It has also been reported that some allosteric effectors-such as chloride ions, inositol hexaphosphate, 2,3-diphospho-glycerate and bezafibrate-considerably lower the oxygen affinity of Hb. In this context, an important question is the extent to which these changes influence the conformational dynamics of the protein. Earlier, we elaborated a challenging method based on phosphorescence quenching, which makes characterizing protein-internal dynamics possible in the ms time range. The experimental technique involves phosphorescence lifetime measurements in thermal equilibrium at varied temperatures from 10 K up to 273 K, based on the signal of Zn-protoporphyrin substituted for the heme in the β-subunits of Hb. The thermal activation of protein dynamics was observed by the enhancement of phosphorescence quenching attributed to O2 diffusion. It was shown that the thermal activation of protein matrix dynamics was clearly distinguishable from the dynamic activation of the aqueous solvent, and was therefore highly specific for the protein. In the present work, the same method was used to study the changes in the parameters of the dynamic activation of human HbA induced by binding allosteric effectors. We interpreted the phenomenon as phase transition between two states. The fitting of this model to lifetime data yielded the change of energy and entropy in the activation process and the quenching rate in the dynamically activated state. The fitted parameters were particularly sensitive to the presence of allosteric effectors and could be interpreted in line with results from earlier experimental studies. The results suggest that allosteric effectors are tightly coupled to the dynamics of the whole protein, and thus underline the importance of global dynamics in the regulation of Hb function.