High-resolution Neutron Structural Analysis of Two States of a Bilin Reductase PcyA

• Project/Area Number: 24570122
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Ibaraki University
• Principal Investigator: UNNO Masaki 茨城大学, 理工学研究科, 教授 (10359549)
• Co-Investigator(Renkei-kenkyūsha): FUKUYAMA Keiichi 大阪大学, 大学院工学研究科, 招へい研究員 (80032283) ; WADA Kei 宮崎大学, テニュアトラック推進機構, テニュアトラック准教授 (80379304) ; TAMADA Taro 原子力機構, 量子ビーム, グループリーダー (50391248)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2014: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 中性子結晶構造解析 / 酵素 / 水素 / 還元反応 / フィコシアノビリン / コンフォメーション / 還元 / 色素 / 光合成 / 水素原子 / ヒドロニウムイオン / J-PARC / ビリン還元酵素 / 反応機構 / 光合成色素 / 光反応 / 中性子結晶解析 / 回折 / 光応答 / プロトネーション

Outline of Final Research Achievements

PcyA catalyzes two of the steps of two-proton-coupled two-electron reduction of biliverdin (BV）, to phycocyanobilin. Revealing the protonation states in the active site of PcyA and the substrate BV is important for understanding this unique reaction mechanism. In the present study, we determined the neutron crystal structure of PcyA in complex with BV. The structure revealed the protonation states of BV and the surrounding residues. We found that two forms of BV, neutral BV and protonated BVH+, were coupled with 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 third example of a hydronium ion in a protein structure. Neutron analysis also revealed how X-ray irradiation of the PcyA-BV crystal altered the structure of the PcyA-BV complex.

Research Products

• [Journal Article] Insights into the Proton Transfer Mechanism of a Bilin Reductase PcyA Following Neutron Crystallography. (2015)
  • Author(s): Unno, M., Ishikawa-Suto, K., Kusaka, K., Tamada, T., Hagiwara, Y., Sugishima, M., Wada, K., Yamada, T., Tomoyori, K., Hosoya, T., Tanaka, I., Niimura, N, Kuroki, R., Inaka, K., Ishihara, M., Fukuyama, K.
  • Journal Title: J. Am. Chem. Soc. Volume: in press Issue: 16 Pages: 5452-5460
  • DOI: 10.1021/jacs.5b00645
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Structures of the substrate-free and the product-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae: X-ray crystallography and molecular dynamics investigation (2013)
  • Author(s): Unno, M., Ardevol, A., Rovira, C., and Ikeda-Saito, M.
  • Journal Title: The Journal of Biological Chemistry Volume: 288 Issue: 48 Pages: 34443-34458
  • DOI: 10.1074/jbc.m113.486936
  • Peer Reviewed
• [Presentation] 中性子結晶構造解析によるフェレドキシン依存性ビリン還元酵素基質複合体の水素化状態可視化 (2015)
  • Author(s): 海野昌喜
  • Organizer: 第3回物構研サイエンスフェスタ・第6回MLFシンポジウム
  • Place of Presentation: つくば
  • Year and Date: 2015-03-18
  • Invited
• [Presentation] Protonation States of a Bilin Reductase PcyA Following Neutron Crystallography (2015)
  • Author(s): Masaki UNNO
  • Organizer: The 2nd Ibaraki University-Kasetsart University Symposium
  • Place of Presentation: 水戸
  • Year and Date: 2015-02-17
  • Invited
• [Presentation] 中性子結晶構造解析によるフェレドキシン依存性ビリン還元酵素－基質複合体の水素化状態可視化 (2014)
  • Author(s): 海野昌喜、須藤久美子、日下勝弘、玉田太郎、萩原義徳、杉島正一、和田啓、山田太郎、石原真樹子、福山恵一
  • Organizer: 平成26年度日本結晶学会年会
  • Place of Presentation: 東京
  • Year and Date: 2014-11-03
• [Presentation] 中性子解析でビリン還元酵素PcyAの特異なプロトン化反応を見る (2014)
  • Author(s): 海野昌喜
  • Organizer: 平成25年度茨城県ビームラインCROSSトライアルユース成果報告会
  • Place of Presentation: 東京
  • Year and Date: 2014-10-22
  • Invited
• [Presentation] フェレドキシン依存性ビリン還元酵素PcyAの中性子結晶構造解析 (2014)
  • Author(s): 海野昌喜
  • Organizer: BioJapan 2014
  • Place of Presentation: 横浜
  • Year and Date: 2014-10-15
• [Presentation] Neutron Structure of Phycocyanobilin:oxidoreductase Complexed with Biliverdin (2014)
  • Author(s): Masaki Unno, Kumiko Ishikawa-Sudo, Katsuhiro Kusaka, Taro Tamada, Yoshinori Hagiwara, Masakazu Sugisima, Kei Wada, Taro Yamada, Katsuaki Tomoyori, Takaaki Hosoya, Ichiro Tanaka, Nobuo Niimura, Ryota Kuroki, Koji Inaka, Makiko Ishihara, and Keiichi Fukuyama
  • Organizer: 23rd Congress and General Assembly of the International Union of Crystallography
  • Place of Presentation: Montreal
  • Year and Date: 2014-08-09
• [Presentation] 中性子結晶構造解析で見るフェレドキシン依存性ビリン還元酵素PcyAの水素化状態 (2014)
  • Author(s): 海野昌喜、須藤久美子、日下勝弘、玉田太郎、萩原義徳、杉島正一、和田啓、山田太郎、石原真樹子、福山恵一
  • Organizer: 第41回生体分子科学討論会
  • Place of Presentation: 福岡
  • Year and Date: 2014-06-07
• [Presentation] フェレドキシン依存性ビリン還元酵素の水素化反応機構を解明するために (2013)
  • Author(s): 海野昌喜
  • Organizer: 第二回iBIX研究会（2013）
  • Place of Presentation: 東海村
• [Presentation] Structural studies of heme oxygenase unravel catalytic reaction mechanism (2013)
  • Author(s): Masaki Unno
  • Organizer: International Symposium of Homogeneous Chemical Reactivity
  • Place of Presentation: 水戸
• [Book] Integrating Approach to Photofunctional Hybrid Materials for Energy and the Enviroment (2013)
  • Author(s): Masaki Unno, Masakazu Sugishima, Kei Wada, Keiichi Fukuyama
  • Total Pages: 173
  • Publisher: Nova Publishers, NY
• [Book] Integrating Approach to Photofunctional Hybrid Materials for Energy and the Environment (2013)
  • Author(s): Masaki UNNO
  • Total Pages: 173
  • Publisher: Nova Science Publishers inc.
• [Remarks] 茨城大学大学院理工学研究科 海野昌喜
  • URL: http://info.ibaraki.ac.jp/Profiles/19/0001860/profile.html
• [Remarks] 海野昌喜研究室
  • URL: http://www.fas.ibaraki.ac.jp/?page_id=827
• [Remarks] 茨城大学研究者総覧
  • URL: http://info.ibaraki.ac.jp/Profiles/19/0001860/profile.html
• [Remarks] 研究室HP
  • URL: http://www.fas.ibaraki.ac.jp/?page_id=827
• [Remarks] 茨城大学フロンティア応用原子科学研究センター 海野研究室
  • URL: http://www.fas.ibaraki.ac.jp/?page_id=827