Research Article: Visualizing the Bohr effect in hemoglobin: neutron structure of equine cyanomethemoglobin in the R state and comparison with human deoxyhemoglobin in the T state

Date Published: July 01, 2016

Publisher: International Union of Crystallography

Author(s): Steven Dajnowicz, Sean Seaver, B. Leif Hanson, S. Zoë Fisher, Paul Langan, Andrey Y. Kovalevsky, Timothy C. Mueser.


The determination of the positions of H/D atoms in equine cyanomethemoglobin by neutron diffraction is described.

Partial Text

Hemoglobin (Hb) is one of the best-characterized macromolecules and is regarded as an ideal model for studies of protein evolution, cooperativity and allostery. In vertebrates, two heterodimers (α1β1:α2β2) comprise the oxygen-transporting functional tetramer contained at high concentration (∼5 mM tetramer, normal mean corpuscular hemoglobin per mean corpuscular volume) inside the red blood cells (RBCs). This containment is crucial since the tightly coupled deoxy tetramer (Kd ≃ 0.01 nM) would dissociate into liganded relaxed (R-state) dimers (Kd ≃ 1 µM) at lower concentrations (Atha & Riggs, 1976 ▸). Binding of oxygen in the lung, driven by the increased partial pressure of oxygen, the tetramer transitions into the fully liganded R state. As RBCs move away from the lungs, the partial pressure of oxygen declines, leading to ligand dissociation, promoted by the stabilizing effect of the deoxy tense (T-state) tetramer. In the tissue capillaries, the RBCs also encounter an increase in the concentration of dissolved CO2 and a corresponding decrease in pH, where hemoglobin assists in the transport of CO2 and excess protons. Several heterotropic allosteric hemoglobin modulators, including H+, CO2 and 2,3-bisphosphoglycerate (2,3-BPG), regulate the reversible binding of O2. The release and uptake of protons by hemoglobin is known as the ‘Bohr effect’ and ionizable residues influenced by this effect are known as ‘Bohr groups’. Perutz and coworkers proposed that salt bridges present in the T state primarily influence the Bohr effect. The salt bridges involve the α N-terminus, Val1(NA1), bridged to the α C-terminus, Arg141(HC3), and the β C-terminus, His146(HC3), bridged to both αLys40(C6) and βAsp94(FG1) (globin fold positions are denoted in parentheses; Perutz et al., 1969 ▸, 1980 ▸; Perutz, 1970 ▸). Multiple studies have implicated additional residues that contribute to the Bohr effect (Berenbrink, 2006 ▸; Lukin & Ho, 2004 ▸). From a structural point of view, the transition from the R state to the T state alters the local environments of potential Bohr groups. As expected, small changes in relative positions shift the pKa values of ionizable residues, altering the proton affinity of a particular Bohr group (Thurlkill et al., 2006 ▸). Despite the numerous theoretical and experimental studies on hemoglobin, the specific changes in the protonation state of potential Bohr groups in the R and T states remains uncertain, as the techniques that have previously been utilized do not provide direct observation of the protonation state.




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