Structural basis of reaction mechanism and proton pathway of [NiFe]-hydrogenase

2023 Fiscal Year Final Research Report

• Project/Area Number: 19H00984
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: University of Hyogo
• Principal Investigator: Higuchi Yoshiki 兵庫県立大学, 理学研究科, 特任教授 (90183574)
• Project Period (FY): 2019-04-01 – 2024-03-31
• Keywords: ヒドロゲナーゼ / 中性子結晶解析 / X線結晶解析 / プロトン経路 / ラマン分光法 / 酵素活性定量法 / 酸素耐性酵素

Outline of Final Research Achievements

Neutron crystallographic analysis of a standard [NiFe]-hydrogenase in the oxidized form was successfully performed. The active site was found to contain a planar square Ni(II). A low-barrier hydrogen bond at the ligand (S) of the proximal Fe-S was found, suggesting that the Fe-S functions as an electron donor when O2 invades. Based on the crystal structure of the CO-bound form of the weakly oxygen-tolerant hydrogenase, a positive correlation between oxygen and CO tolerance was proposed.
FT-IR analysis of the oxidized NAD+-reducing [NiFe]-hydrogenase was successfully performed, and it was concluded that the Ni(IV) structure of the active site contributes to oxygen tolerance. X-ray analysis of the formate dehydrogenase from one of the plant symbiotic bacteria revealed its evolutionary relationship with other energy-metabolizing enzymes.
We also developed a Raman method to assay hydrogenases, and found that the enzyme is catalytically active mainly at the gas-liquid interface.

Free Research Field

構造生物学

Academic Significance and Societal Importance of the Research Achievements

本研究における学術的意義は，『高分解能中性子結晶解析』による巨大タンパク質内で重要な役割を担う水素原子の可視化であった．元素の中で最も小さな水素原子は，『プロトン，ヒドリド，水素分子』と変幻自在に形を変えて化学反応に関与する．[NiFe]ヒドロゲナーゼの水素分解・合成反応では『ヒドリド』と『プロトン』の挙動が反応機構解明に重要である．特に水分子を含んだ水素結合ネットワークに支えられた高効率プロトン伝達機構の正確な知見を中性子結晶解析法で得ることで，触媒反応を効率化する『タンパク質場』を真に理解できる．この理解が進めば，今後，燃料電池等にも応用して卓越した新規の製品開発に貢献できると考える．