I. Xanthorhodopsin於可見光區的光迴圈效率與波長之相依性研究  II. 從動力學與熱力學觀點探討紫膜中細菌視紫質暗適應過程之溶劑同位素效應

第一篇 Xanthorhodopsin (xR)為鑲嵌於嗜鹽真桿菌Salinibacter ruber細胞膜上之光合成蛋白。xR的反應中心與其他視紫質蛋白質不同，除了視黃醛分子之外，尚有另一個類胡蘿蔔素分子Salinixanthin (SX)作為視黃醛的天線，延伸短波長光吸收範圍，而SX吸收的光子能量會傳遞至視黃醛上使xR進行光迴圈反應，此特性為目前所知生物體內最簡單的能量傳遞系統。本實驗將使用可調變激發波長之時間解析差異吸收光譜，觀察xR之M態瞬態濃度與其激發波長相依性，進而獲得能量傳遞效率，並討論SX吸收接近紫外光區較高能量光子的能量傳遞過程。以570 nm直接激發xR視黃醛吸收峰，可得到最高的光迴圈效率。吾人以此波長量得的M態瞬態濃度為基準，將其他波長與之比較，計算相對M態量子產率。激發波長520–430 nm區域中，越靠近短波長處視黃醛吸收越少，光迴圈所需能量逐漸轉變為由SX吸收光子並進行能量傳遞而提供，因此量子產率隨波長變短而遞減。其中，視黃醛在430 nm處幾乎已無吸收，故在此激發波長下所得之光迴圈效率可視為單獨激發SX的結果。從文獻得知430 nm光子能量足以由雙光子方式激發細菌視紫質(bacterorhopdopsin, bR)視黃醛之S2能階並進行後續光迴圈反應，但在xR系統內，激發波長430 nm與460 nm所得之M態量子產率兩者皆為37%左右，表示兩激發波長下所偵測之M中間態皆為視黃醛S1能階之光迴圈產物，激發態之SX不會直接傳遞能量至視黃醛S2能階並進行其後的光迴圈反應。第二篇細菌視紫質為嗜鹽古生菌Halobacterium salinarum細胞膜上之光合成蛋白，光激發其中的化學分子視黃醛可使其進行光異構化，引發其光迴圈反應及質子幫浦而形成膜內外側的氫離子梯度，可做為合成ATP之化學勢位。除了光異構化反應外，視黃醛也會進行由all-trans、15-anti至13-cis、15-syn之熱異構化反應，意即暗適應態的生成。過去研究中指出此反應具有溶劑反同位素效應，但其涉及之動力學與熱力學現象並未充分討論。故本實驗將使用溶劑H2O及D2O，由紫外可見吸收光譜法觀察以紫膜形式存在之細菌視紫質的視黃醛吸收峰在溫度30˚C—55˚C內隨時間變化之吸收光譜。吾人發現在此溫度區間內，反應溫度低於45˚C時具有反同位素效應，即kf (D2O) / kf (H2O) > 1。經由建立反應模型並以過渡態理論分析實驗數據，得到視黃醛分子在H2O及D2O環境中由基態至過渡態的反應焓ΔH*f分別為24.7±1.2與20.1±0.4 kcal mol–1，而相對的反應熵ΔΔS*f = ΔS*f (D2O) − ΔS*f (H2O)為–14.4±3.9 cal mol–1 K–1，配合文獻研究可計算得D2O環境中吉布斯自由能低於H2O環境0.4—0.7 kcal mol-1。在此研究結果表示在熱異構化反應中，相較於反應物，分子處於過渡態時，其結構中具有較強的氫鍵。

關鍵字

光迴圈效率；波長相依性；能量傳遞；細菌視紫質；溶劑同位素效應；暗適應過程

並列摘要

Part I. Xanthorhodopsin (xR) is a dual-chromophore proton-pump photosynthetic protein comprising one retinal Schiff base and one light-harvesting antenna salinixanthin (SX). The excitation wavelength-dependent transient population of the intermediate M demonstrates that the excitation of the retinal at 570 nm leads to the highest photocycle activity and the excitations of SX at 460 and 430 nm reduce the activity to ca. 37% relatively, suggesting an energy transfer pathway from the S2 state of the SX to the S1 state of the retinal and a quick internal vibrational relaxation in the S2 state of SX prior to the energy transfer from SX to retinal. Part II. The thermal retinal isomerization from all-trans, 15-anti to 13-cis, 15-syn of bacteriorhodopsin in purple membrane in H2O and D2O during dark adaptation was investigated at 30—55°C at neutral pH. In this temperature range, phase transition of purple membrane and destruction of the tertiary structure of bacteriorhodopsin did not take place. We found that the solvent isotope effect is inverted below about 45°C; i.e., kf (D2O) / kf (H2O) > 1. Applying the transition state theory, the changes in enthalpy from the initial state to the transition state along the thermal trans-to-cis forward reaction coordinate, ΔH*f, were determined to be 24.7 ± 1.2 and 20.1 ± 0.4 kcal mol−1 in H2O and D2O, respectively. The relative entropic change of the transition state in H2O and D2O, ΔΔS*f = ΔS*f (D2O) − ΔS*f (H2O), was −14.4 ± 3.9 cal mol−1 K−1. In addition, the Gibbs free energy of trans-to-cis thermal isomerization reaction in D2O is 0.4—0.7 kcal mol−1 lower than that in H2O. It is the first time the entropy and enthalpy of the transition state have been quantified to elucidate the solvent isotope effect in the retinal thermal isomerization of bacteriorhodopsin during dark adaptation. The solvent isotope effect on the thermodynamics properties and kinetics implied that the hydrogen bonding in the transition state during the dark adaptation of bR is stronger than that in the initial state.

並列關鍵字

xanthorhodopsin ； phtotcycle activity ； energy transfer ； bacteriorhodopsin ； solvent isotope effect ； dark adaptation

參考文獻

(5)Balashov, S. P.; Imasheva, E. S.; Boichenko, V. A.; Antón, J.; Wang, J. M.; Lanyi, J. K. Science 2005, 309, 2061–2064.

(15)Imasheva, E. S.; Balashov, S. P.; Wang, J. M.; Lanyi, J. K. Photochem. Photobiol. 2006, 82, 1406–1413.

(17)Boichenko, V. A.; Wang, J. M.; Antón, J.; Lanyi, J. K.; Balashov, S. P. Biochim. Biophys. Acta 2006, 1757, 1649–1656.

(18)Balashov, S. P.; Imasheva, E. S.; Wang, J. M.; Lanyi, J. K. Biophys. J. 2008, 95, 2402–2414.

(7)Balashov, S. P.; Imasheva, E. S.; Boichenko, V. A.; Antón, J.; Wang, J. M.; Lanyi, J. K. Science 2005, 309, 2061–2064.

國際替代計量

I. Xanthorhodopsin於可見光區的光迴圈效率與波長之相依性研究 II. 從動力學與熱力學觀點探討紫膜中細菌視紫質暗適應過程之溶劑同位素效應

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