Functional analysis of hemoglobin molecules locked in doubly liganded conformations

A controversy still exists over whether the molecular basis of hemoglobin cooperativity can be more appropriately explained by one of two classic allosteric models, the concerted and sequential models. To distinguish these two models from the viewpoint of their fundamental processes, namely, the presence or absence of conformational equilibria, we have trapped the conformations of nickel(II)-iron(II) hybrid hemoglobin molecules with two CO-bound, alpha2(Fe-CO)beta2(Ni) and alpha2(Ni)beta2(Fe-CO), by encapsulation in the water-filled pores of sol-gel-derived transparent silica-gels. In our experimental system, nickel(II) protoporphyrin binds neither O2 nor CO and mimics a fixed deoxyheme, and the gel matrix provides a means of inhibiting large-scale protein structural changes, thus enabling O2 equilibrium study of the hybrids still in their doubly liganded conformations. Results showed that two conformations of widely different O2 affinity exist together in each doubly liganded hemoglobin, providing a direct proof of the concerted mechanism versus the sequential mechanism.