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

2014 Fiscal Year Final Research Report

• 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
• 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

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.

Free Research Field

構造生物化学