Photon Factory Activity Report 2022 #40 (2023)

BL-5A/2013G122, 2017G033

High-resolution structural studies of NADH-cytochrome b5 reductase
Yu HIRANO1,2, Shigenobu KIMURA3, Kunio MIKI4 and Taro TAMADA1,*1National Institutes for Quantum Science and Technology, Inage, Chiba 263-8555, Japan
2The Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
3Ibaraki University, Hitachi, Ibaraki, 316-8511, Japan

4Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

1 Introduction
Many redox proteins utilize cofactors such as flavinadenine dinucleotide (FAD) and nicotinamide
adenine dinucleotide (NADH) to conduct proton andhydride transfer reactions. Precise structural
information including hydrogen atoms is required tounderstand the molecular mechanisms underlying
the redox reactions. NADH-cytochrome b5 reductase(b5R) has a role for preserving cellular redox balanceand functions as an electron supplier for various
metabolic processes. The redox reaction cycle of b5R
includes the proton and hydride transfer steps via
NADH and FAD cofactors [1]. We performed highresolution structure analyses of porcine liver b5R byboth X-ray and neutron crystallography to reveal thestructural basis of the redox reaction [2]. Because Xray diffraction data generally contain higher resolution
structural information about heavy atoms (those otherthan hydrogen atoms), the combination with neutron
diffraction data is expected to provide more precise
information about atomic positions involvinghydrogen atoms bound to heavy atoms.
2 Experiment
Crystallization experiments were performed by thesitting-drop vapor diffusion method under ananaerobic condition. Large crystals of the oxidized
forms of b5R with the sizes of approximately 2.0 mm3
were obtained in two pD (potential of deuterium)
conditions at pD 6.5 and 7.5.
The X-ray diffraction data sets for joint Xray/neutron refinement were collected with thecrystals used for neutron diffraction data collection at
BL-5A beamline. The wavelengths of X-rays were set
to 0.75 Å (pD 6.5) and 0.76 Å (pD 7.5). A joint Xray/neutron structure refinement was performed withthe program Phenix [3].
3 Results and Discussion
The X-ray and neutron diffraction data sets wereobtained at 0.88 Å (pD 6.5) and 0.87 Å (pD 7.5)
resolutions (Table 1). Hydrogen (H) and deuterium(D) atoms were included in the model during the Xray/neutron joint refinement. Neutron scattering
lengths are negative for H (-0.37×10-12 cm) andpositive for D (0.67×10-12 cm). Thus, neutronscattering-length-density map can differentiate H andD atom positions. The neutron Fo-Fc H/D omit map
calculated at 1.45 Å (pD 6.5) and 1.50 Å (pD 7.5)

resolutions clearly shows all H/D atom positions
bound to FAD (Fig. 1) and the H/D atom positions of
the amino acid residues and solvent molecules
involved in hydrogen-bonds with FAD for both pDconditions.
A structural difference between pD 6.5 and pD 7.5
was observed for the hydrogen-bond network from
FAD to the protein surface. The side chain of His49
which is located in the hydrogen-bond network is in
the double-protonated state at pD 6.5 and in the
single-protonated state at pD 7.5. In addition, thestopped-flow UV-visible spectroscopy suggested thata histidine residue is involved in the b5R redox
reaction cycle. The structural and spectroscopic
studies indicated that the hydrogen-bond network isthe proton pathway in b5R.
Table 1: Diffraction data statistics
pD 6.5pD 7.550-0.88
50-0.87
Resolution (Å)(0.90-0.88)(0.89-0.87)
Unit cell a, b, c
48.6, 72.2,48.7, 72.3,
(Å)
85.1
85.1
Completeness100.0 (99.9)
96.2 (91.7)(%)
Rsym (%)
6.9 (43.0)
5.6 (44.7)
68.4 (4.0)
55.0 (2.7)
I/σ(I)

Fig. 1: Fo-Fc neutron-scattering-length-density omit
map of FAD.
Acknowledgement
We thank Drs. Kazuo Kurihara, Katsuhiro Kusaka
and Andreas Ostermann for the neutron diffraction
data and the PF beamline staff for the X-ray
diffraction data.

Photon Factory Activity Report 2022 #40 (2023)

References[1] M. Yamada et al., JMB 425, 4295 (2013).
[2] Y. Hirano et al., submitted.
[3] D. Liebschner et al., Acta D 75, 861 (2019).
* tamada.taro@qst.go.jp