Structural origin of cooperativity in human hemoglobin: a view from different roles of α and β subunits in the α2β2 tetramer

Abstract

This mini-review, mainly based on our resonance Raman studies on the structural origin of cooperative O₂ binding in human adult hemoglobin (HbA), aims to answering why HbA is a tetramer consisting of two α and two β subunits. Here, we focus on the Fe-His bond, the sole coordination bond connecting heme to a globin. The Fe-His stretching frequencies reflect the O₂ affinity and also the magnitude of strain imposed through globin by inter-subunit interactions, which is the origin of cooperativity. Cooperativity was first explained by Monod, Wyman, and Changeux, referred to as the MWC theory, but later explained by the two tertiary states (TTS) theory. Here, we related the higher-order structures of globin observed mainly by vibrational spectroscopy to the MWC theory. It became clear from the recent spectroscopic studies, X-ray crystallographic analysis, and mutagenesis experiments that the Fe-His bonds exhibit different roles between the α and β subunits. The absence of the Fe-His bond in the α subunit in some mutant and artificial Hbs inhibits T to R quaternary structural change upon O₂ binding. However, its absence from the β subunit in mutant and artificial Hbs simply enhances the O₂ affinity of the α subunit. Accordingly, the inter-subunit interactions between α and β subunits are nonsymmetric but substantial for HbA to perform cooperative O₂ binding.

Keywords: Cooperativity; Hemoglobin; Iron-histidine bond; Quaternary structure; Resonance Raman; Subunits.

Fig. 1

a Molecular structure of human adult hemoglobin (HbA); red, α subunit; blue, β subunit from 2DN2 (Park et al. 2006). b The molecular structure of heme (Fe-protoporphyrin IX complex)

Fig. 2

Illustrative diagram of hybrid Hbs. The circle denotes the α (no color) or β (gray) subunits, comprising Fe (or Ni) heme, with Fe presence coordinated by the His residue of a protein. a α(Fe²⁺)β(Fe³⁺: M²⁺) An O₂ or CO molecule can bind to the Fe²⁺ heme of the α subunit. b α(Fe³⁺: M²⁺)β(Fe²⁺) An O₂ or CO molecule can bind to the Fe²⁺ heme of the β subunit (M represents a metal like Ni)

Fig. 3

a UVRR spectra of deoxyHbA (A) and COHbA (B) at pH 6.7 excited at 235 nm and their difference (C) [= (A) − (B)]. Reprinted (adapted) with permission from Nagatomo et al. (2011a), Fig. 2. Copyright (2011) American Chemical Society. b Newly defined spectrum (S), which is the average of the spectra of deoxyHbA and COHbA. It shows spectrum (A). The digitally obtained difference spectra of the deoxyHbA-spectrum (S) and COHbA-spectrum (S) are delineated by traces (B) and (C), respectively, which indicate the states of the 100%T and 100%R in the difference spectra. Reprinted (adapted) with permission from Nagatomo et al. (2011a), Fig. 6. Copyright (2011) American Chemical Society

Fig. 4

Illustrative representation of Hb quaternary structures in various liganded states. The left and right half-circles symbolically represent the α1 and β2 subunits, respectively, and the two squares between them indicate the Tyr (Y) and Trp (W) residues at the subunit contact region. The squares in blue and yellow indicate the T- and R-contacts respectively, and their areas are proportional to the relative numbers with T and R contact. Light blue and pink indicate the nearly T- and R-contacts, respectively. L denotes CO molecules. His in a rectangle denotes the proximal (F8) histidine. The heme shape is represented by a bar (planar) or bowl (domed), and the Fe-His bond is represented, if present, by a short line between the heme and His. The solid lines and waved lines under the His indicate the constrained or relaxed states of the Fe-His bond, respectively. These Hbs could be categorized into “T,” “intermediate,” and “R” structure based on the color distribution of the contact region. When the colors of both Y and W are blue and yellow, the corresponding Hbs are categorized as “T” and “R,” respectively. Hbs with mixed colors are designated into an “intermediate” group. Reprinted (adapted) with permission from Nagatomo et al. (2011a), Fig. 10. Copyright (2011) American Chemical Society

Fig. 5

Oxygen equilibrium curves of human adult hemoglobin (HbA) (black) and cavity mutant Hb, rHb(βH92G) (blue). The insertions illustrate the relation between the F helix and F8 histidine. Source: Nagatomo et al. . Molecular structure of HbA is from 2DN2 (Park et al. 2006) and that of rHb(βH92G) is shown by modification from 2DN2 (Park et al. 2006)

Fig. 6

Amino acid residues potentially involved when the relaxation of the F helix of the β subunit is communicated to the α subunit. This pathway was deduced with ultraviolet resonance Raman spectroscopy (UVRR)

Fig. 7

Raman spectra, A~D, of the α(Fe²⁺) (left panel) and β(Fe²⁺) (right panel) subunits. In both, the spectra, A, at the top were observed for isolated chains. The second spectra, B, were observed for valency hybrid tetramers, the α(Fe²⁺)β(Fe³⁺) (left panel) and α(Fe³⁺)β(Fe²⁺) (right panel) placed in the R state. The third spectra, C, were observed for the α(Fe²⁺)β(Fe³⁺) (left panel) and α(Fe³⁺)β(Fe²⁺) (right panel) placed in the T state. The spectra, D, at the bottom were observed for the deoxyHbM Milwaukee at pH 6.5 (left panel) and deoxyHbM Boston at pH 6.5 (right panel). Source: Nagai K, Kitagawa T, 1980

Fig. 8

O₂ binding equilibrium curves of HbA and structures of individual subunits in the tetramer. The cleavage of the Fe-His bond of the β subunit increases the affinity of the α subunit but, in contrast, that of the α subunit does not increase the affinity of the β subunit and does not induce a quaternary structure change (T → R)

Fig. 9

Demonstration of the Fe-His stretching RR band asymmetry of the α subunit in deoxyHbMs Hyde Park and Milwaukee, [α(Fe²⁺)β(Fe³⁺)]. The two deconvoluted bands in the T structure are shown by a red line. The blue-colored bands, which appear at higher pH, are attributed to the Fe-His stretching RR band of the R structure. Reprinted (adapted) with permission from Nagatomo et al. (2017b), Fig. 6. Copyright (2017) American Chemical Society