葉綠體之H+-ATP合成脢活性動力學之研究

Abstract

ATP合成脢為一負責合成ATP之膜蛋白，存在於細菌、葉綠體和粒線體中。菠菜的ATP合成脢可以利用葉綠體最內層膜的氫離子濃度梯度或電位能差來合成ATP。我們的研究主要分成兩大主題：一是討論pH值對菠菜的ATP合成脢的活性的影響；另一是討論ATP合成脢的臨界行為。 為了討論膜內外絕對pH值對菠菜ATP合成脢的活性的影響，我們設計一連串固定氫離子濃度梯度的實驗，以確認其活性是否單純與濃度梯度有關。為了描述與解釋實驗結果，我們提出結合relay groups的概念以建立新的動力學模型、蛋白質序列比對與量子化學計算。綜合其實驗與理論結果我們針對pH值對菠菜ATP合成脢活性的影響提出一新的分子層次的動力學模型。 為了觀察菠菜ATP合成脢的臨界行為，我們設計了兩組實驗參數：一是溫度參數、另一是鈣離子濃度。我們發現在溫度攝氏40度附近，ATP合成脢的活性會驟然提高。另外，膜內鈣離子濃度也會影響ATP合成脢的活性，且隨著實驗溫度不同，活性變化也不同。根據此實驗觀察，我們建立一generalized Langevin equation，理論模型中加入因臨界行為造成的與時間關聯性的熱擾動，發現在臨界現象的區間，活性會驟然提高。

H+-ATP synthase is an integral membrane enzyme, which catalyzes ATP synthesis in bacteria, chloroplasts and mitochondria. In chloroplasts of spinach, ATP synthesis can be driven by proton concentration difference pH, and electric potential  across the membrane. Our research could be divided into two distinct topics. One is the study for pH-dependent activity behavior of CFoF1, and the other one is the study for the critical-like behavior of CFoF1. To elucidate the pH-dependent activity behavior of CFoF1, we proposed a molecular kinetic model consisting of two proton relay groups, each being characterized with a specific proton dissociation constant, pK. From appropriately designed experiments, the pK values of the relay groups were extracted to be 5.1 for pKin and 8.3 for pKout, respectively. By aligning the subunit a of spinach with that of E.coli., we identified the candidate residues that may play the role of the proton relay groups: aAsp-197 and aGlu-198 for input relay groups, and aGlu-178, aAsp-179 and aLys-182 for output. We adapted the quasichemical theory of solvation to calculate the theoretical pK values of the candidate residues, and identified the most plausible candidates of the proton relay groups. Overall, a molecular kinetic mechanism for protons conducting through CFoF1 during ATP synthesis and its physical implications on the specific reactivity behavior of CFoF1 are presented. To observe the critical-like behavior of CFoF1, we performed the activity experiments with two factors, temperature and calcium ion, respectively, which were reported to be capable of inducing critical-like behavior of lipid bilayers. We found that at around 40℃, the activity of CFoF1, reconstituted in PC/PA 19:1 liposomes critically increased 2 times of the room temperature one. To observe the Ca++ effect, we prepared the CFoF1-reconsituted liposomes with various internal Ca++ concentrations to measure the activity of ATP synthesis. We found that at around [Ca++] = 2.5 mM, the activity seemed to have critical increase, and this Ca++ concentration happened to be located at the critical-like region of the pure liposomes. To explain our experimental observation with critical fluctuation, we establish a generalized Langevin equation, which is incorporated with the critical correlation time, to simulate the rotational movement of CFo. We found that with long correlation time, the rotational frequency increased 2.3-4.5 times of the one with short correlation time. This indicates that the critical fluctuation of the membrane could enhance the rotational movement of CFo.