An Investigation of the Distal Histidyl Hydrogen Bonds in Oxyhemoglobin: Effects of Temperature, pH, and Inositol Hexaphosphate

Abstract

On the basis of X-ray crystal structures and electron paramagnetic resonance (EPR) measurements, it has been inferred that the O2 binding to hemoglobin is stabilized by the hydrogen bonds between the oxygen ligands and the distal histidines. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO2 A) in solution. Here, the NMR spectra of uniformly 15N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH and temperature, and also in the presence of an organic phosphate, inositol hexaphosphate (IHP). We have found significant effects of pH and temperature on the strength of the H-bond markers, i.e., the cross peaks for the side-chains of the two distal histidyl residues, α58His and β63His, which form H-bonds with the O2 ligands. At lower pH and/or higher temperature, the side-chains of the distal histidines appear to be more mobile and the exchange with water molecules in the distal heme pockets is faster. These changes in the stability of the H-bonds with pH and temperature are consistent with the changes in the O2 affinity of Hb as a function of pH and temperature and are clearly illustrated by our NMR experiments. Our NMR results have also confirmed that this H-bond in the β-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. IHP has only a minor effect on these H-bond markers compared to the effects of pH and temperature. These H-bonds are sensitive to mutations in the distal heme pockets, but not affected directly by the mutations in the quaternary interfaces, i.e., α1β1 and/or α1β2 subunit interface. These findings provide new insights regarding the roles of temperature, hydrogen ion, and organic phosphate in modulating the structure and function of hemoglobin in solution.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

“…In the center of the cavity are the proximal F8His and the distal E7His. F8His covalently binds the heme onto the protein for O 2 binding, whereas E7His stabilizes the bound O 2 with hydrogen bonds (52,53) and acts as a gate for ligand entry (52,54).…”

New Look at Hemoglobin Allostery

“…At higher pH and/or lower temperature, the cross-peaks for both α58His ( 1 Hε 2 , 15 Nε 2 ) and β63His ( 1 Hε 2 , 15 Nε 2 ) are clearly visible in Figure 15 (taken from Figure 2 of ref. 131 ). When pH is decreased and/or at higher temperature, the side-chains of the distal histidines appear to be more mobile, and the exchange with water molecules in the distal heme-pockets is faster.…”

“…It is of interest to note that the H-bond between the bound oxygen and E7His in the β-subunit is weaker than that in the α-subunit, 131 while the β-subunit has also a slower k ox . Therefore, the stereo-placement of amino acid residues in the heme-pocket is the major contributing factor in determining the k ox of the subunit.…”

“…Through the use of naturally occurring human mutant hemoglobins as well as of a large number of recombinant mutant Hbs produced by using our Hb expression plasmid, we have carried out an extensive array of structural, dynamic, kinetic, and functional studies. 67,81,86,90,96,120,131,138,153,156,168-176 Some of the observed structural, dynamic, kinetic and functional changes can be related to the mutations. Some of them can be small, but the mutation at one site could affect the conformation of distant as well as neighboring residues.…”

WAXS Studies of the Structural Diversity of Hemoglobin in Solution

“…Molecular dynamics (MD) studies indicate increased fluctuations of the α-helices in deoxy-Hb compared to the liganded structures (16) and it has been proposed that these fluctuations control the coordination mode of the heme Fe with the proximal and distal histidine thereby regulating oxygen affinity (16). NMR studies have detailed clear differences in the dynamics of the deoxy- and liganded-forms of hemoglobin (17–19) and increased mobility of the side-chains of the distal histidines has been directly correlated with decreased oxygen affinity (20). …”