Photon Factory Activity Report 2019 #37 (2020)

BL-NE3A/2019G104

X-ray diffraction experiment for joint X-ray/neutron structure determination ofCuNIR

Yohta FUKUDA1, Yu HIRANO2, Tsuyoshi INOUE1, Taro TAMADA2,*
1 Osaka University, Suita, Osaka, 565-0871, Japan
2 National Institutes for Quantum and Radiological Science and Technology, Tokai, Ibaraki,319-1106, Japan
1 Introduction
Many enzymes of microorganisms play importantroles for the nitrogen cycle on the earth. Atmosphericnitrogen gas is converted into chemical species,
including ammonia and nitrate, by the nitrogen
fixation reaction in microorganisms. The reduction ofnitrate in the soil to nitrogen gas is performed by the
microbial denitrification process. Copper-containing
nitrite reductase (CuNIR) catalyze the reduction ofnitrite to nitric oxide, which is a key step in the
denitrification process. CuNIR has one type 1 copper(T1Cu), which is the electron acceptor fromphysiological partner proteins, and has one type 2copper (T2Cu), which is involved in the nitrite
reduction. To understand the molecular mechanismof the nitrite reduction, we have determined neutroncrystal structure of CuNIR from Geobacillus
thermodenitrificans (GtNIR).
2 Experiment
Crystals with a volume more than 1 mm3 areusually required for neutron diffraction experiment. A
large crystal of GtNIR was prepared by a seeding
method. Seed crystals of GtNIR were obtained by thehanging-drop vapor-diffusion method at 293 K. The
reservoir solution contained polyethylene glycol 4000
as a precipitant. A seed crystal was transferred to alarge volume (150 μl) of crystal growth solution
consisting of equal volume of protein solution and thereservoir solution. After a large-volume crystal wasobtained, the reservoir solution was exchanged todeuterated cryo-protectant solution. Deuterium (2H)
reduces background intensities in neutron diffraction
data collection compared to hydrogen (1H). The
crystal was cooled in a nitrogen-gas stream at 100 K
for neutron and X-ray diffraction data collection.
Neutron diffraction data set was collected at theBL03 beamline (iBIX) of J-PARC/MLF. After theneutron diffraction experiment, X-ray diffraction dataset was collected using the same crystal at the ARNE3A beamline of PF-AR. The wavelength of X-rays
was set to 1.00 Å and diffraction intensities weremeasured with Pilatus 2M-F detector. The crystal
structure of GtNIR was determined by the molecularreplacement method with the crystal structure of the same
protein as a search model (PDB ID: 4YSO). The joint Xray/neutron (XN) refinement was performed using thePhenix program.

3 Results and Discussion
The X-ray diffraction data set was obtained at 1.03
Å resolution (Table 1). The crystal belongs to thespace group R3 (H3) with unit cell parameters of a =114.2 Å, b = 114.2 Å, c = 83.7 Å in H3 setting. The joint
X/N refinement was performed with the X-ray data up to1.3 Å resolution because R-factors were improved andelectron density map showed less noises with theresolution limit. The final model consists of 4952 atoms in
the protein molecules and 458 atoms of water molecules
including H/D atoms and Rwork/Rfree values for the X-ray
data were converged to 9.62/11.1%.
The crystal structure analysis clearly indicated thatthe molecular species coordinating the T2Cu is adeuterated hydroxide (OD-) molecule. The energetic
preference of the coordination of a hydroxide waspredicted by the DFT calculation in the nitrite
reduction of GtNIR [2]. The hydrogen-bond network,
which is connecting the T1Cu and T2Cu sites, forms
a stable H-bond deducing from a H/D exchange ratioof the histidine residue (His134) located at thenetwork. The H-bond network has been proposed toconsist of a part of the intramolecular electrontransfer pathway in CuNIR [3].
Table 1: X-ray diffraction data statistics
Resolution (Å)
42.6-1.03 (1.05-1.03)
Space group
R3 (H3)114.2, 114.2, 83.7
Unit cell a, b, c (Å)(H3)
Completeness (%)
99.9 (98.7)
Rmerge (%)
6.5 (64.1)<I/σ(I)>
21.2 (3.5)

Fig. 1: Active site of GtNIR

Photon Factory Activity Report 2019 #37 (2020)

Acknowledgement
We thank the PF beamline staff for data collection.
References[1] Y. Fukuda et al., Proc. Natl. Acad. Sci. USA 117,4071 (2020).
[2] M. Lintuluoto & J.M. Lintuluoto, Biochemistry, 55,
210 (2016).
[3] R.G. Hadt et al., J. Am. Chem. Soc., 136, 15034(2014).
* tamada.taro@qst.go.jp