Photon Factory Activity Report 2018 #36 (2019)

BL-1A, BL-17A/2016G012

Structural basis of the oxygen dissociation mechanism of the giant hemoglobinin the presence of the allosteric effectors
Nobutaka NUMOTO
Medical Research Institute, Tokyo Medical and Dental University (TMDU),
Bunkyo-ku, Tokyo 113-8510, Japan*

1 Introduction
Allosteric oxygen-binding property of hemoglobin (Hb)
is well studied for the past a half century. The molecularmechanism of the cooperative binding of oxygen
molecules to each subunit has been understood based on
the crystal structures of the fully-oxygenated (oxy) anddeoxygenated (deoxy) forms of mammalian Hbs [1].
However, it remains to be clarified structures of oxy-deoxy
intermediate forms without any artificial modification ofthe Hb molecule.
We have reported that an extracellular giant Hb form
marine tube worm, Lamellibrachia satsuma is able to shift
from the oxy to deoxy form in keeping the crystalline state[2]. However, Hb of L. satsuma show little susceptibility
for allosteric effectors such as calcium or magnesium ions.
To elucidate the effects for cooperative oxygen-binding
mechanism for the giant Hbs, we have performed thecrystal structure analyses of the oxy-deoxy intermediate
forms of the giant Hb from another marine worm,
Oligobrachia mashikoi (Oligobrachia Hb), which show
significant enhancement under existence of calcium ion.
2 Experiment
Oligobrachia Hb was prepared and crystallized as oxy
form as described [3]. The oxy-deoxy intermediate crystals
were obtained from the oxy crystals through the soaking
method. The crystals were transferred to the cryoprotectant
solution containing PEG 400 in a step-wise manner,increasing to a final concentration of 20% (v/v). In the laststep, the crystals were soaked in the cryoprotectant
solution containing 50-100 mM sodium hydrosulfite. The
soaking time for the final solution was varied from 10 to120 sec., and then immediately flash-frozen in a nitrogengas stream at 95 K. Oxygen saturation fraction wasmeasured by absorption spectra of each crystal.
X-ray diffraction experiments were performed atbeamelines 1A and 17A at PF, KEK. The data wereprocessed and scaled using XDS [4] and were truncatedusing the CCP4 program suite [5]. The structure weresolved by the molecular replacement method using
MOLREP [6]. Several cycles of manual model rebuilding
and refinement were performed by using the program
COOT [7] and REFMAC5 [8], respectively.
3 Results and Discussion
When soaking the oxy-crystals in the final solution, it
was observed that the color of the crystals graduallychanged from bright red to deep purple under microscopeobservation. However, due to variations in crystal volume,the correlation between the soaking time and the oxygen
saturation fraction will be weak. Therefore, the oxygen
saturation fraction of individual crystals was identified by
microspectroscopy. Partial oxygen saturation fractions
were also confirmed by the obtained electron density forthe bound oxygen at the heme pocket, because the electron
densities are clearly weaken.
Comparisons of the oxy-structure with the intermediate
structure revealed that local structural changes correlatewith the large quaternary changes. For example, the side
chain conformation of Arg residue near the heme (Figure1) is clearly associated with the quaternary structuralchanges. The electron densities for the calcium ions arevery weak or disappeared for the oxy-deoxy intermediate
which have deoxygenated quaternary structure. These
results confirm our previous estimation [9] that the calcium
ions bound to the interface between subunits play a keyrole for the allosteric transition of the structure of the giant
Hbs.

Fig. 1: Comparison between the fully-oxygenated (left)
and 63% oxygenated structure (right). The quaternary
structures are that of oxy, and deoxy forms, respectively.
References[1] M. F. Perutz, Nature 228, 726 (1970).
[2] N. Numoto et al., Acta Cryst. D70, 1823 (2014).
[3] N. Numoto et al., PNAS 102, 14521 (2005).
[4] W. Kabsh, Acta Cryst. D66, 125 (2010).
[5] M. D. Winn et al., Acta Cryst. D67, 235 (2011).
[6] A. Vagin and A. Teplyakov, Acta Cryst. D66, 22 (2010).
[7] P. Emsley et al., Acta Cryst. D66, 486 (2010).
[8] G. N. Murshudov et al., Acta Cryst. D67, 355 (2011).
[9] N. Numoto et al., Biochemistry 47, 11231 (2008).
* numoto.str@mri.tmd.ac.jp