Assessment of Roles of Surface Histidyl Residues in the Molecular Basis of the Bohr Effect and of β143 Histidine in the Binding of 2,3-Bisphosphoglycerate in Human Normal Adult Hemoglobin

Abstract

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges

“…Specifically, the golden-mantled ground squirrel, thirteenlined ground squirrel, Uinta chipmunk and least chipmunk share an unusual substitution at the C-terminus of the α-chain, α141Arg→Cys, and the major β-chain of the golden-mantled ground squirrel has an unusual substitution at the C-terminus, β146His→Gln (Fig.3). Both C-terminal residues are known to contribute to the pH sensitivity of Hb-O 2 binding (the Bohr effect) in human Hb (Perutz, 1970;Weber and Fago, 2004;Perutz, 1983;Berenbrink, 2006;Fang et al, 1999;Lukin and Ho, 2004). The golden-mantled ground squirrel also has a highly unusual substitution at another highly conserved β-chain position, β55Met→Ile (Fig.3).…”

“…With respect to the Bohr effect, studies of human HbA have demonstrated that H + ions mainly reduce Hb-O 2 affinity by protonating the α-amino termini and the C-terminal β146His, which stabilizes the deoxy T-state through the formation of additional salt bridges within and between subunits (Berenbrink, 2006;Fang et al, 1999;Lukin and Ho, 2004;Perutz, 1970;Perutz, 1983;Weber and Fago, 2004). The C-terminal α141Arg does not directly contribute to the Bohr effect [the pK a (acid dissociation constant) of Arg is 12.0, so it is overwhelmingly protonated at pH7.4], but it contributes indirectly to the Cl --dependent Bohr effect by facilitating the Cl --assisted charge stabilization of the free-NH 2 terminus of α1Val.…”

“…Accordingly, human HbA mutants that have the same residues as the golden-mantled ground squirrel (Hb Nunobiki, α141Arg→Cys and Hb Kodaira, β146His→Gln) are characterized by a marked reduction in the alkaline Bohr effect (Harano et al, 1992;Kwiatkowski and Noble, 1987;Shimasaki, 1985). In the case of the β-chain carboxy terminus, the positive charge on β146His is stabilized in the T-state by the negative charge of β94Asp and, at least in human HbA, it has been estimated that the oxygenationlinked deprotonation of β146His accounts for most of the Bohr effect (Fang et al, 1999;Lukin and Ho, 2004;Perutz, 1983;Perutz et al, 1985). In Hb Kodaira (and presumably in the major isoHb of the golden-mantled ground squirrel that also possesses β146Gln), replacement of the β94-β146 salt bridge by an unionizable H-bond would theoretically result in a diminished Bohr effect.…”

Positive allosteric modulators to peptide GPCRs: a promising class of drugs

“…The key examples of allosteric modulator characterization are the detailed studies on aspartic transcarbamylase and its feedback inhibition [4,5] . Other examples of allosteric actions involve non-enzymatic proteins such as the oxygen carrier hemoglobin, whose affinity is regulated by protons through the Bohr effect without involving binding to the O 2 site, or by 2,3-bisphosphoglycerate binding to an allosteric site on hemoglobin that affects O 2 trapping [8,9] .…”

Theoretical study of interactions between human adult hemoglobin and acetate ion by polarizable force field and fragmentation quantum chemistry methods

“…According to the mechanism proposed by Perutz [2] , the cooperative effects arise from the equilibrium between two alternative quaternary states, the deoxy T (or low-affinity) state and the oxy R (or highaffinity) state. In addition to CO 2 , Cl − , and HPO 4 2− , the concentrations of 2,3-biphosphoglycerate (2,3-BPG) will affect the oxygen affinity [3][4][5][6][7] . 2,3-BPG is a small, highly anionic organic phosphate synthesized in red blood cells (RBCs).…”