Photon Factory Activity Report 2014 #32 (2015)
BL-5A, AR-NW12A, AR-NE3A/2013G504

Neutron and X-ray Crystal Analysis of a Bilin Reductase PcyA
Masaki Unno1, 2*, Katsuhiro Kusaka2, Taro Tamada3, Yoshinori Hagiwara4, Masakazu Sugishima5,
Kei Wada6, and Keiichi Fukuyama 7, 81
Graduate School of Science and Engineering, Ibaraki University,
4-12-1 Nakanarusawa, Hitachi, Ibaraki 316-8511, Japan
2
Frontier Research Center for Applied Atomic Sciences, Ibaraki University,
162-1 Shirakata, Tokai, Naka, Ibaraki 319-1106, Japan
3Quantum Beam Science Center, Japan Atomic Energy Agency,
2-4 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1195, Japan
4Department of Biochemistry and Applied Chemistry, Kurume National College of Technology,
1-1-1 Komorino, Kurume, Fukuoka 830-8555, Japan
5
Department of Medical Biochemistry, Kurume University School of Medicine,Kurume, Fukuoka 830-0011, Japan
6
Organization for Promotion of Tenure Track, University of Miyazaki,5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan
7
Graduate School of Science, Osaka University,
1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan,
8
Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanWe determined the neutron crystal structure of PcyA in complex with BV. This is the first study of the neutronstructure of a ferredoxin-dependent bilin reductase (FDBR) family member, revealing the protonation state of BV
and the surrounding residues. We found that two forms of BV, neutral BV and protonated BVH+, were coupledwith the two conformation/protonation states of the essential residue Asp105. Further, His88 and His74 near BV
were singly protonated and were connected with an intervening hydronium ion. To our knowledge, this is the thirdexample of a hydronium ion in a protein structure. Neutron analysis also revealed how X-ray irradiation of thePcyA-BV crystal altered the structure of the PcyA-BV complex. It should be noted that the present structure
provides a structural basis for the reaction mechanisms of other FDBRs.
1 Introduction
Bilin compounds are fundamentally important foroxygenic photosynthetic organisms because they areutilized as pigments for photosynthesis (phycobilins) andphotoreceptors (phytochromobilin). Red algae andcyanobacteria use phycobilisomes, complexes comprisedof phycobiliproteins, to harvest photon energy, and theseorganisms adapt to light quality and nutrient conditions
by changing the components of the phycobilisome.
Phycocyanobilin:ferredoxinoxidoreductase(PcyA)
catalyzes two of the steps of two-proton-coupled twoelectron reduction of biliverdin IXα (BV), a heme
catabolite, to phycocyanobilin. Revealing the protonation
states in the active site of PcyA and the substrate BV is
important for understanding this unique reaction
mechanism; however, previous structural analyses wereunsuccessful in determining the location of hydrogenatoms in PcyA because X-rays are not strongly diffracted
by hydrogen atoms.
2 Experiment2.1. Crystallization
The crystallization procedure and conditions wereperformed to grow the large crystals necessary forneutron crystallography as follows: Purified PcyA wasconcentrated to 120 mg/mL by centrifugation using an
Amicon Ultra centrifugal. The PcyA-BV complex wasprepared by incubating the concentrated PcyA and BV
overnight on ice. The crystal for neutron diffraction wasobtained by the sitting drop vapor diffusion method at293 K from a 20 µL drop of the PcyA-BV complexsolution and 20 µL of the reservoir solution containing
200 mM NaCl, 1.4 M ammonium sulfate, and 50 mM
MES (pH 5.9). These procedures were performed underdark conditions except for a small green spotlight. The
crystal was grown to approximately 2.5 mm × 1.8 mm ×0.6 mm in 1 month.
2.2. Neutron diffraction experiment. The crystal wassoaked in 50 µL of 50 mM MES buffer (pD 6.3)
containing 200 mM NaCl and 1.4 M ammonium-d8
sulfate (D8, 98%; Cambridge Isotope Laboratories Inc.)prior to neutron diffraction. The soaking solution was
exchanged three times in 3 weeks. Time-of-flight (TOF)
neutron diffraction data were collected at BL03 iBIX at
the Materials and Life Sciences Experimental Facility(MLF) of the Japan Proton Accelerator ResearchComplex (J-PARC, Tokai, Japan) at room temperature

Photon Factory Activity Report 2014 #32 (2015)

under dark conditions. A total of 24 data sets werecollected using a wavelength of 3.0 to 5.6 Å with adetector distance of 490 mm. Exposure time for eachdata set was 9 hours at 300 kW. The TOF neutron data
were indexed, integrated, scaled, and processed withSTARGazer. The same crystal was used for X-ray
diffraction experiment at room temperature.
2.3. X-ray diffraction experiment. X-ray diffraction
data from the same crystal were collected using an ADSC
Quantum270 CCD detector at NE3A at room temperature.
The wavelength of the synchrotron radiation,transmittance, and slit size were 1.0 Å, 10%, and 0.05
mm (vertical) × 0.05 mm (horizontal), respectively. The
sample-to-detector distance and oscillation range were150.7 mm and 1.0°, respectively. In an effort to minimize
the amount of damage due to radiation, the position of thecrystal during irradiation was changed for each shot.
Data were integrated, merged, and processed with HKL2000 software.
2.4. X-ray intensity data, structure, and absorption
spectra from independent crystals with high-dose andlow-dose synchrotron irradiation at 100 K. The
crystals were soaked in a crystallization solution
containing 25% (v/v) glycerol, and flash frozen in liquid
N2. X-ray intensity data were collected at 100 K usingsynchrotron radiation (λ = 1.0 Å) from BL5A. The
oscillation angle, exposure time, slit size, and sample-todetector distance were 1º/frame, 1.0 sec, 0.2 mm (v) × 0.2mm (h), and 175.8 mm, respectively. X-rays wereattenuated by the insertion of an aluminum filter. The
transmittances of the X-rays were 30% (thickness of Al =0.3 mm) and 1.0% (thickness of Al = 1.2 mm) for highand low-dose data, respectively. The number of images
in each data set was 135.
3 Results and Discussion
In this study, we succeeded in obtaining a very large
crystal of the PcyA-BV complex and soaked it into thedeuterated solution to exchange the hydrogen atoms in
the crystal with deuterium atoms. As a result, neutron
diffraction intensity data were able to be collected. We
have determined the neutron crystal structure of thePcyA-BV complex at room temperature at a 1.95 Åresolution.
The wild-type PcyA-BV structure, by
avoiding X-ray-induced photo reduction, also revealed
the existence of the axial water molecule. These results,
including hydrogen localizations, will provide crucial
information for elucidating the unique catalytic
mechanisms of PcyA [1].
We found two protonation states in the PcyA-BV
complex. One is BVH+ and Asp105-, in which four
pyrroles of BV were protonated and Asp105 wasdeprotonated. The other is the combination of the neutral
BV and neutral Asp105. In this state, an axial water ishydrogen-bonded to the BV A-ring pyrrole.
His88 Nδ was protonated to form a hydrogen bondwith the lactam O atom of the BV A-ring. His88 and
His74 were linked by hydrogen bonds via H3O+. These
results imply that Asp105, His88, and the axial watermolecule contribute to proton transfer during PcyA
catalysis.
At first, we modeled the water molecule on theneutron-scattering length density map between His88 andHis74 in a similar manner to a previous X-ray
crystallographic study [2]. We found residual neutronscattering length densities close to the His88 Nδ and Nεatoms. Nδ-protonated His88 was nicely fitted to theresidual density, whereas the residual density near theNε atom was significantly far enough from the Nε atom
to be modeled as a hydrogen atom. The density is out of
the imidazole plane, although a proton bound to theNε atom should be in the plane. From these facts, thedensity close to His88 Nε was not likely corresponded tothe H atom covalently bound to the His88 Nε atom. The
water molecule was regarded as a hydronium ion(actually D3O+) (Fig. 1).

Fig. 1: A hydronium ion intervening His88 and His74
Acknowledgement
We thank members of iBIX at J-PARC for neutron
diffraction data collection. The neutron diffraction
experiment was conducted under proposal 2012PX0011(Ibaraki Prefecture Project). We thank Dr. Yusuke
Yamada and other members of the Structural Biology
beamlines of PF and members of BL41XU of SPring-8for X-ray diffraction data collection including preliminaryexperiments. The X-ray diffraction experiments in PF
were conducted under proposals 2011G519 and2013G504.
Crystallization under microgravity wasattempted in the “Kibo” project of the Japan Aerospace
Exploration Agency (JAXA).
We are grateful toProfessor Takamitsu Kohzuma of Ibaraki University forhis support and fruitful discussions regarding
crystallization. We also thank Professor Masao IkedaSaito’s laboratory at Tohoku University for use of themicrospectrophotometer. This work was partly supportedby JSPS KAKENHIs numbers 24570122, 22770096 (toM.U.), and 23370052 (to K.F., K.W., and M.U.), andInnovation Research Promotion Program of Ibaraki Univ.
(to M.U. and K.K.).
References[1] M. Unno et al., J. Am. Chem. Soc. 137, 5452 (2015).
[2] Y. Hagiwara et al., Proc. Natl. Acad. Sci. U.S.A.,

103, 27-32, (2006)

Research Achievements
1. This work was broadcasted on NHK Mito local
news.
* unno19@mx.ibaraki.ac.jp