Contribution of surface histidyl residues in the alpha-chain to the Bohr effect of human normal adult hemoglobin: roles of global electrostatic effects.
We have applied site-directed mutagenesis to our Escherichia coli hemoglobin expression plasmid and constructed five recombinant mutant hemoglobins (r Hbs): r Hb(alpha20His-->Gln or alpha:H20Q); r Hb(alpha:H50Q); r Hb(alpha:H72Q); r Hb(alpha:H89Q); and r Hb(alpha:H112Q). We have constructed these r Hbs to help us assess the contribution of the surface histidyl residues in the alpha-chain to the alkaline Bohr effect of human normal adult hemoglobin (Hb A). In our laboratory, we have monitored the variation of proton nuclear magnetic resonances arising from the C2 protons of the histidyl residues of Hb A as a function of pH and buffer conditions. Several of these resonances have been assigned to the individual histidyl residues on the surface of the hemoglobin molecule using naturally occurring mutant hemoglobins and chemically modified hemoglobins. In the present work, we have identified the C2 proton resonances of five surface histidyl residues of the alpha-chain, alpha20, alpha50, alpha72, alpha89, and alpha112, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance spectra of Hb A with those of the r Hbs. For the assignment of the C2 proton resonances of alpha20His and alpha112His, we have used combinations of mutations to compensate for the spectral perturbations resulting from the single mutations, which obscure the resonance assignment. On the basis of the new findings, in solvent containing 0.1 M chloride, the overall contributions from surface histidyl residues of both the alpha- and beta-chain and from other previously identified alkaline Bohr groups account for approximately 75% of the observed Bohr effect at pH 7.3 (the maximum Bohr effect under the prescribed solvent conditions). Our results show that some histidyl residues contribute to the Bohr effect and some oppose the net Bohr effect. In some cases, the addition of anions can diminish or reverse the contributions of specific histidyl residues to the overall Bohr effect. Thus, the Bohr effect, a heterotropic effect, depends on the intricate arrangement and interactions of all hydrogen and anion binding sites in the hemoglobin molecule. It is an excellent example of global electrostatic effects in proteins.
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Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Zou
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Ho
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D000328:Adult; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics; D005914:Globins / Q000737:chemistry* / Q000235:genetics; D006441:Hemoglobin A / Q000737:chemistry / Q000235:genetics; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D006454:Hemoglobins; D011994:Recombinant Proteins; D006639:Histidine; D005914:Globins; D006441:Hemoglobin A; D010100:Oxygen