Experimental resolution of cooperative free energies for the ten ligation states of human hemoglobin.

Abstract

Tetrameric human hemoglobin can assume ten molecular forms that differ in the number and configuration of ligands bound at the four heme sites. For each of these species we have determined the cooperative free energy-i.e., the deviation in free energy of ligation from that which would obtain for the same sites binding as independent a and f8 subunits. These cooperative free energies were resolved from measurements on the dissociation into dimers of tetramers in which each subunit is either unligated (Fe2' deoxy) or "ligated" by conversion into the cyanomet form (Fe31 CN). The results indicate that each hemoglobin tetramer acts as a three-level molecular switch. During the course of ligation, the total cooperative free energy (6 kcal/mol over all four binding steps) is expended in two transitions that are synchronized with particular ligation steps. Whether a cooperative energy transition occurs or not depends upon how the ligation step changes both the number and configuration of ligated subunits. The hemoglobin tetramer is thus a "combinatorial switch." The finding of three distinct free energy levels for the ten ligation states suggests the existence of three major structural forms of the hemoglobin tetramer.

Multiple strategies for O2 transport: from simplicity to complexity

“…This observation, which is consistent with the possibility of a manifold T quaternary state, lead to the formulation of an extension of the MWC model (105), which allowed to account in a quantitative fashion for some of the most problematic challenges for the MWC model (106,107). In particular, this model postulates the existence of a cooperative unit (called 'cooperon'), within which the interaction among active sites is modulated by direct interactions; the original MWC model operates only among different cooperons which form the whole oligomeric protein.…”

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals

“…The solution oxygen binding curves contain relatively little information, and numerous models have been proposed over the years that are consistent with such data (Antonini & Bmnori, 1971;Imai, 1982;Wyman & Gill, 1990). The investigation of tetramerdimer dissociation equilibria has focused on artificial intermedi-ates, particularly those containing the cyanide complex of oxidized hemes (Smith & Ackers, 1985;Perrella et al, 1990a;. This work has led to the conclusion that the twostate MWC model is incompatible with the experimental results (Ackers & Smith, 1987;Ackers, 1990;Ackers et al, 1992).…”

The Contribution of the Asymmetric α1β1 Half-oxygenated Intermediate to Human Hemoglobin Cooperativity

“…The original evidence for this preferred ligation pathway was the hyper (170-fold) stability of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) relative to the other three doubly liganded species (7)(8)(9). However, this evidence has been proven to be an artifact arising from valency exchange between the deoxy and cyanometheme sites during the long deoxy incubation that had been used routinely in the laboratory of Ackers (11,12).…”

“…In 1985, this methodology was first applied to resolution of the tetramer stabilities of all ten ligation intermediates of the deoxy-cyanomet [Fe(II)/ Fe(III)-CN Ϫ ] hybrid system, in which cyanometheme mimics a fixed oxyheme (8). The most striking finding was that the dimer-tetramer association equilibrium constant for (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) was about 170 times that of the other three doubly liganded species at pH 7.4, which implies a hypercooperativity in specific ligation steps in the ␣1␤1 dimer within the tetramer (i.e.…”