Roles of the .beta.146 histidyl residue in the molecular basis of the Bohr effect of hemoglobin: a proton nuclear magnetic resonance study

Abstract

Assessment of the roles of the carboxyl-terminal beta 146 histidyl residues in the alkaline Bohr effect in human normal adult hemoglobin by high-resolution proton nuclear magnetic resonance spectroscopy requires assignment of the resonances corresponding to these residues. Previous resonance assignments in low ionic strength buffers for the beta 146 histidyl residue in the carbonmonoxy form of hemoglobin have been controversial [see Ho and Russu (1987) Biochemistry 26, 6299-6305; and references therein]. By a careful spectroscopic study of human normal adult hemoglobin, enzymatically prepared des(His146 beta)-hemoglobin, and the mutant hemoglobins Cowtown (beta 146His----Leu) and York (beta 146His----Pro), we have resolved some of these conflicting results. By a close incremental variation of pH over a wide range in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer, a single resonance has been found to be consistently missing in the proton nuclear magnetic resonance spectra of these hemoglobin variants. The spectra of each of these variants show additional perturbations; therefore, the assignment has been confirmed by an incremental titration of buffer conditions to benchmark conditions, i.e., 0.2 M phosphate, where the assignment of this resonance is unambiguous. The strategy of incremental titration of buffer conditions also allows extension of this resonance assignment to spectra taken in 0.1 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane buffer. Participation of the beta 146 histidyl residues in the Bohr effect has been calculated from the pK values determined for the assigned resonances in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer. Our results indicate that the contribution of the beta 146 histidyl residues is 0.52 H+/hemoglobin tetramer at pH 7.6, markedly less than the 0.8 H+/hemoglobin tetramer estimated by study of the mutant hemoglobin Cowtown (beta 146His----Leu) by Shih and Perutz [(1987) J. Mol. Biol. 195, 419-422]. We have found that at least two histidyl residues in the carbonmonoxy form of this mutant have pK values that are perturbed, and we suggest that these pK differences may in part account for this discrepancy. Furthermore, summation of the positive contribution of the beta 146 histidyl residues and the negative contribution of the beta 2 histidyl residues to the maximum Bohr effect measured in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer suggests that additional sites in the hemoglobin molecule account for proton release upon ligation greater than the contribution of the beta 146 histidyl residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Inversion of the Bohr effect upon oxygen binding to 24-meric tarantula hemocyanin.

“…This movement leads to a change in the distances between the conserved histidineglutamate pairs, which might well result in changes of the pK value of the imidazole ring of His-239, in analogy to the breaking of the salt bridge between His-146,8 and Asp-94P in hemoglobin (36,37). In hemoglobin, His-146f3 adopts only two different pK values during oxygen binding.…”

Direct Determination of Protonation States of Histidine Residues in a 2 Å Neutron Structure of Deoxy-Human Normal Adult Hemoglobin and Implications for the Bohr Effect

“…6 Based on X-ray crystallographic analyses, Perutz 7 first proposed αVal1 and βHis146 as possible Bohr groups; they form salt bridges with αArg141 and βAsp94 in the T state, which break on transition to the R state, thus changing their pK a . However, results from several experiments [8][9][10][11][12][13][14] and also theoretical calculations [15][16][17] indicate that a large number of residues contribute in either a positive or a negative way to the overall Bohr effect and that the exact roles of these residues change under different conditions.…”

Novel Mechanisms of pH Sensitivity in Tuna Hemoglobin

“…The "alkaline Bohr effect" plays an important role in modifying the oxygen affinity of Hb to increase oxygen uptake in the lungs and delivery in the tissues. A large body of research, occasionally marked by strident disagreements, is devoted to relating this effect to Hb structure (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Hb exists in two principal forms, the T state and the R state, which are the preferred conformations of the protein in the absence and presence of heme ligands, respectively.…”