Assessment of roles of surface histidyl residues in the molecular basis ofthe Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(beta H116Q) and rHb(beta H143S). Purified rHbs wer e used to assign the C2 proton resonances of beta 116His and beta 143His an d to resolve the ambiguous assignments made over the past years. In the pre sent work, we have identified the C2 proton resonances of two surface histi dyl residues of the beta chain, beta 116His and beta 143His, in both the ca rbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resona nce (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHb s. Current assignments plus other previous assignments complete the assignm ents for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measure d in deuterium oxide (D2O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethan e 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, beta 146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta 143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha 20His, alpha 112His, and beta 117His have essentially no contr ibution; alpha 50His, alpha 72His, alpha 89His, beta 97His, and beta 116His have moderate positive contributions; and beta 2His and beta 77His have a moderate negative contribution to the Bohr effect. The sum of the contribut ions from 24 surface histidyl residues accounted for 86% of the alkaline Bo hr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Althou gh beta 143His 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, beta 143His is not essential for the binding of 2,3-BPG in the neutr al pH range according to the proton NMR and oxygen affinity studies present ed here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr eff ect microscopically for novel recombinant Hbs with important functional pro perties, such as low oxygen affinity and high cooperativity. The present st udy further confirms the importance of a global electrostatic network in re gulating the Bohr effect of the hemoglobin molecule.