Perutz has proposed that the cooperative effect of oxygen binding in tetrameric hemoglobin arises from an equilibration between two quaternary structures: the liganded or relaxed (R) structure with oxygen affinity comparable to the isolated subunit affinity and the unliganded, tensed (T) structure with oxygen affinity lowered by constraining salt bridges [1–3]. Perutz argues that the equilibrium between these two structures is primarily governed by displacement of the iron and proximal histidine from the mean porphyrin plane and that much of the free energy of heme–heme interaction is stored in salt bridges which break upon oxygenation. This theory has received support from other workers [4–6]. On the other hand EXAFS studies have shown that ironpyrol nitrogen bond distances do not differ between deoxyhemoglobin A and deoxyhemoglobin Kempsey (β99 asp → asn) which is a high affinity mutant essentially devoid of cooperativity [7]. Thermodynamic studies of the temperature dependence of αβdimer–tetramer equilibrium point out that the free energy of heme–heme interaction is stored in hydrogen bonds between dimers and not salt bridges [8]. Replacement of iron by cobalt, which remains low spin even in the unliganded tetramer, only slightly diminishes n, the empirical measure of cooperativity [9–]. Finally, the displacement of iron from the porphyrin plane in deoxyhemoglobin A seems to differ little from iron displacement in the noncooperative monomer deoxymyoglobin [12]. We wish to propose a truly alternative mechanism for cooperative ligand binding by hemoglobin which does not necessitate metal movement or salt bridge energetics. The important molecular movement for an increase in oxygen affinity is porphyrin sliding from the hydrophobic protein interior to a position with increased porphyrin exposure to solvent. In hemoglobin A, this movement is rigidly coupled to breaking the hydrogen bond between β99 asp and α42 tyr as the porphyrin moves towards the protein exterior, and upon oxygenation formation of a new hydrogen bond between β102 asn and α94 asp. Protein crystallographic studies report that the pophyrins of both α and β subunits are more exposed to water in the met-form than for deoxyhemoglobin [2]. The porphyrin is more exposed to solvent in the β chain of the high affinity mutant deoxyhemoglobin Yakima (β99 asp → his) than A [13]. Studies on model cobaltoporphyrins report that oxygen affinity is more dependent upon porphyrin–solvent interactions than upon 2,4-substituents or ligand trans to oxygen [14, 15]. Stellwagen has pointed out that the redox potential of hemoproteins is dependent upon the degree of porphyrin exposure to solvent, with the redox potential decreasing as porphyrin exposure increases [16]. That oxygen affinity increases with increasing porphyrin exposure to solvent is consistent with all the above facts and is tied together by the experiments of Basolo and co-workers who have shown that an inverse linear correlation exists between th logK of oxygenation and cobalt(II/III) redox potential for a series of equatorially substituted cobalt complexes [17, 18]. Recent work upon energetics across the α 1β 2 interface of hemoglobin shows that amino acid mutations in this region drastically alter cooperative energetics while substitutions at other parts of the molecule have little effect upon cooperativity [19]. Substitution of β99 asp by his, gln or gly destroys the lone hydrogen bond which connects the deoxy-dimers and abolishes all cooperative energy, and the resultant hemoglobins have oxygen affinities close to those of isolated subunits. The NMR resonance position of this hydrogen bond (−14.2 ppm) is in the range of resonance positions of ‘strong’ hydrogen bonds while the resonance position of the β102 asn−α94 asp proton (−10.3 ppm) appears normal [10, 21]. So energy storage for the deoxy tetramer is primarily localized in this β99 asp−α42 tyr hydrogen bond. Information between subunits is transferred through those amino acids involved in hydrogen bonding to heme pyrrole II and the innate porphyrin rigidity used to modulate porphyrin exposure to solvent which in turn controls oxygen affinity. Porphyrin sliding can also account for Feimidazole bond rupture in hemoglobin NO in the presence of IHP [22].
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Jun 1, 1983
D L Rousseau + 1
Open Access