Conformational changes in an ultrafast lightdriven enzyme determine catalytic activity Olga A. Sytina , Derren J. Heyes , C. Neil Hunter , Maxime T. Alexandre , Ivo H. M. van Stokkum , Rienk van Grondelle & Marie Louise Groot Nature 456, 1001-1004 (18 December 2008) Miyasaka Lab. Yuji MORII Contents • Introduction Catalyst, Enzyme Light-driven enzyme • Experiments • Results and discussion • Summary Catalyst （触媒） Catalyst Products Reactants ・increase the rate of a reaction ・high yield Gibbs energy (a) Uncatalyzed ⊿‡G (b) Catalyzed reaction ⊿ｒG○ Reactants Products Extent of reaction Enzyme （酵素） Candida rugosa lipase （カンジダ・ルゴサのリパーゼ） Enzymes are biomolecules that catalyze chemical reactions. Alomost all enzymes are proteins Ref. Protein Data Bank Light harvesting antenna （光捕集アンテナ） Ｌｉｇｈｔ R N Mg N H R N N N Mg H H N ？ MeO2C CO2H N N H MeO2C CO2H Pchlide Chlide (プロトクロロフィリド) (クロロフィリド) The more light is exposed to the enzyme, the more the reaction advances. Sunny day, more Chlide is biosynthesized Protochlorophyllide oxidoreductase （プロトクロロフィリド酸化還元酵素） Active site Pchlide （プロトクロロフィリド） NADPH Photoreaction at the active site （活性中心で行われる光反応） Hydride transfer ： NADPH → C17 (Pchlide) Proton transfer ： Tyr189 (POR) → C18 (Pchlide) Ref. J.Bio.Chem. 281, 26847-26853 (2004) Absorption spectrum （吸収スペクトル） Incident light Transmitted light (入射光) (透過光) I I’ Sample E Excited State Ground State Absorbance I Abs log ( I I ' ) I' 0 Wavelength Experiments t Probe Detector Sample Pump [Visible pump-probe experiments] 600 ～ 700 nm Detect ・Transient absorption ・Stimulated emission ・Bleached absorption I0 Abs(t ) log I (t ) Transient absorption (過渡吸収スペクトル) R N N Mg N H R N N Mg H H N N N H MeO2C CO2H Pchlide MeO2C CO2H Chlide Question • Under weak light condition Low yield R N N H H N H N Mg Mg N R N N N H MeO2C CO2H MeO2C CO2H • Under strong light condition High yield R N Mg N H R N N N Mg N H H N H MeO2C CO2H MeO2C CO2H N Special set-up ～Lissajous sample scanner～ probe pump Sample cell Each part of the sample was illuminated by a single shot of the laser Results ～Visible pump-probe experiments ～ Excitation cycle 1,2 Region Ⅰ The signal from the Pchlide substrate Blue shift 6-12 Region Ⅱ The signal from the Chlide-precursor species (I675*) 26-55 Depend upon the scan number Inactive enzyme (触媒不活性な状態の酵素) Blue shift diagram 【Scan number : 1,2】 No signal Enzymes do not catalyse Active enzyme (触媒活性な状態の酵素) Blue shift and I675* signal diagram 【Scan number : 26-55】 dependent upon the scan number Photon turns the enzyme ‘on’ Summary [the results from the femtosecond visible pump-probe] Absorption of the first photon activates the enzyme, which results in a high quantum yield formation of products when a second photon is absorbed. First photon Inactive Conformation change Second photon Catalytic reaction Active
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