Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state.

Nobutaka Numoto,Seiki Baba,Nobutoshi Ito,Yoshihiro Fukumori,Yoshiaki Kawano,Kunio Miki,Hideo Okumura

doi:10.1107/s2052252521009386

Abstract

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now. Here, intermediate forms of the 400 kDa giant Hb from the tubeworm Oligobrachia mashikoi are reported. To overcome the above problems without any artificial modifications to the protein or prosthetic groups, intermediate crystals of the giant Hb were prepared from fully oxygenated crystals by a soaking method. The oxygen saturation of the crystals was measured by in situ observation with a microspectrophotometer using thin plate crystals processed by an ultraviolet laser to avoid saturation of absorption. The oxygen saturation of each subunit was determined by occupancy refinement of the bound oxygen based on ambient temperature factors. The obtained structures reveal the detailed relationship between the structural transition and oxygen dissociation. The dimer subassembly of the giant Hb shows strong correlation with the local structural changes at the heme pockets. Although some local ternary-structural changes occur in the early stages of the structural transition, the associated global ternary-structural and quaternary-structural changes might arise at about 50% oxygen saturation. The models based on coarse snapshots of the allosteric transition support the conventional two-state model of Hbs and provide the missing pieces of the intermediate structures that are required for full understanding of the allosteric nature of Hbs in detail.

Highlights

The mechanism of cooperative oxygen binding of hemoglobin (Hb) was explained about half a century ago by Perutz’s stereochemical model (Perutz, 1970) based on the crystal structures of oxygenated Hb and deoxygenated Hb and the theoretical Monod–Wyman–Changeux (MWC) model (Monod et al, 1965) for multi-subunit allosteric proteins
The oxygen saturation fraction of the crystals was calculated by fitting a linear combination of the reference absorption spectra for the oxy
The observed oxygen saturation of the giant Hb from another annelid, Lammelibrachia satsuma, can processed crystals was 100–50% in most cases, and few crystals be made to shift to the deoxy form while maintaining the with under 50% oxygen saturation could be obtained

Summary

Introduction

The mechanism of cooperative oxygen binding of hemoglobin (Hb) was explained about half a century ago by Perutz’s stereochemical model (Perutz, 1970) based on the crystal structures of oxygenated (oxy) Hb and deoxygenated (deoxy) Hb and the theoretical Monod–Wyman–Changeux (MWC) model (Monod et al, 1965) for multi-subunit allosteric proteins. A series of large conformational changes were reported in detail, but because the ligand saturation rate was artificially restrained, there is still room for exploration with regard to how the structural changes occur at other ligandsaturation rates. Another example of the intermediate structure has been reported for an invertebrate homodimeric Hb from the clam Scapharca inaequivalvis (Knapp et al, 2006, 2009; Nienhaus et al, 2007). The ligand saturation was changed continuously and reversibly in the crystalline state (Knapp et al, 2006); no large quaternary-structural change was observed, small changes in ternary structure and hydrogenbond networks at the interface of the dimer structure were thought to play an essential role in the cooperative mechanism

Methods

Results

Conclusion