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

Abstract

Although cooperative oxygenation of human adult hemoglobin (HbA) 1 has been studied extensively as a paradigm for regulatory actions of allosteric proteins, the functional and structural properties of its eight partially oxygenated intermediates remain elusive. This is mostly due to the strong cooperativity of Hb, which suppresses relative abundance of the intermediates, precluding direct study. One of the most specific methods for studying the oxygenation intermediates has been to substitute the heme in one or more of four subunits with another metalloprotoporphyrin IX, which does not bind O 2 while mimicking either normal deoxyheme or oxyheme (1-6). However, there has been a limitation to this approach: Six asymmetric forms cannot be studied in isolation, because the asymmetric hybrid tetramers dissociate into dimers, which then reassociate to form not only the former asymmetric tetramers but also symmetric ones (i.e. dimer rearrangement).Ackers and his colleagues (7) have partly circumvented this difficulty by measuring the dimer-tetramer equilibrium constants for asymmetric hybrid species in the presence of the symmetric parental species. 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. a 170-fold affinity change). Subsequently, the effects of pH, temperature, and single-site mutations on the dimer-tetramer equilibrium of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) were studied (7, 9, 10). It was suggested that this key intermediate assumes a form of the deoxy-Tquaternary structure as judged from the nature of the dimerdimer interface.Based on the hyperthermodynamic stability and T quaternary structure of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤), Ackers' group proposed a new framework for Hb cooperativity, called a symmetry rule (SR), in 1991 (9). The key features of the SR model are: (i) The two ligation steps leading to the asymmetric ␣1␤1 half-liganded intermediate show distinctly favorable cooperativity while those leading to the other three doubly liganded intermediates exhibit no favorable cooperativity; (ii) The asymmetric ␣1␤1 half-liganded intermediate assumes a deoxy-T-quaternary structure while the other three doubly liganded intermediates exhibit an oxy-R-quaternary structure.Recently, we showed that both the published hyperstability and T structure assignment for (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) were artifacts 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). Ackers et al. (13) have now accepted the valency exchange artifacts in their previous studies using de...

Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models

“…The cooperon model could explain significant cooperativity found by Ackers and co-workers for the T quaternary structure obtained indirectly and with assumptions from tetramer-dimer dissociation experiments (51,52). However, direct measurements of oxygen binding (18,53) showed these to be incorrect. The cooperon model also cannot explain the finding of subunits with the same affinity in both quaternary structures.…”

Reverse Engineering the Cooperative Machinery of Human Hemoglobin

“…Another study disagrees with the strength of such effect [16]. Nevertheless, the first two oxygens tend to bind to one dimer rather than distribute to both dimers [6], [17].…”

“…3), since such singly ligated dimer presents a jammed lever system at the dimeric core, thus costs extra energy to form [17]. The key disagreement here is whether the second binding event to form two singly ligated dimers is barely cooperative [16] or negatively cooperative [17]. Despite any potential experimental errors, the molecular code of Ackers does not discard MWC but reflects the molecular mechanism of symmetry conservation of MWC.…”

Evolution of allosteric models for hemoglobin

“…Analysis of more recent tetramer-dimer dissociation experiments of Ackers and coworkers, using chemical analogues that much more closely retain the properties of unaltered hemoglobin (14), have indicated that d T is as small as 4 (33). However, the results of these experiments have been called into question by Morimoto and coworkers (47), whose kinetic measurements lead to an even smaller value for d T .…”