菠菜中葉綠體之H+ -ATP合成脢的活性和結構之研究

Abstract

生物體內的ATP合成脢負責將光合作用或氧化過程所獲得的能量轉換成以ATP的形式儲存◦至今已知ATP 合成的驅動力主要為質子，在少數物種則為鈉離子跨過膜所產生的電化學梯度◦而在葉綠體的體系中٫目前只發現驅動力來自質子的梯度◦ 在本研究，我們利用化學發光的技術探討鈉離子和鋰離子是否能驅動ATP的合成? 結果發現在葉綠體的體系中， 鈉離子也能驅動ATP的合成其初速度為3.5 s-1雖然比質子驅動的初速度~200 s-1慢，但是卻是比本身以鈉離子驅動的菌種(Propionigenium modestrum)的初速度0.7 s-1快 。至於鋰離子，本身不會驅動ATP的合成但是因著有區域性水解(localized hydrolysis)而釋放質子所以當外加140 mV的電場就能合成ATP，其初速度為13.2 s-1。我們也探討鈉離子或鋰離子和質子同位相和不同位相 (從脂質體的角度)時對H+ -ATP合成脢初速度的影響其結果發現: 當不同位相時，鈉離子不會影響質子驅動的ATP合成速率無論是ΔpH 4.0或是ΔpH 3.3; 但是，鋰離子卻是會阻斷CF0的H+ 通道並減慢以質子驅動的合成速率在ΔpH 3.3 時；但是當ΔpH 4.0時，鋰離子的效應不明顯◦ 另一面，當質子和鈉離子或鋰離子同位相時，鈉離子會和質子競爭CF0中的質子通道(ΔpH 4.0)而鋰離子則會阻斷質子通道而使ATP合成的初速度降低(ΔpH 3.5) ◦ 另外٫ 我們也藉著動態光散射光譜來研究H+ -ATP合成脢中親水性亞基CF1 和親脂性亞基CF0之間的作用力◦ 研究結果發現無論是氧化態或是還原態的H+ -ATP合成脢CF1 和CF0之間的作用力皆為離子間的作用力並且在結合成CF0F1的過程中並無水分子的參與◦藉著計算解離常數和熱力學函數其結果顯示氧化態比還原態的H+ -ATP 合成脢更為穩定◦ 因著ATP合成脢的重要性，已過有數以百計的論文以大腸桿菌和粒線體為主角來研究其結構； 其主要結構可以分為兩部分，分別是親水性的F1(進行ATP 合成或水解的反應區域) 和親脂性的F0( 質子或鈉離子通過產生離子梯度以提供化學能) ◦ 但是因著葉綠體的體系和動物細菌的體系不同٫ 並且在結構的次單元上也有些許的差異， 所以我們利用單分子能量共振技術(single molecule fluorescence resonance energy transfer) 來研究葉綠體H+-ATP合成脢在空間上相對結構的問題◦ 我們將染料(TMR 和 Cy5)分別上在b1 和ε的結構次單元上並將整顆的H+-ATP合成脢種在脂質體上 ，藉著觀察能量轉移的效率推算出空間上兩個次單元的相對距離分別為5.3 nm，6.6 nm和 7.6 nm。其ε次單元是位在旋轉的軸承上而b1次單元是位在旁邊用以連接CF1和CF0。其實驗結果和文獻上大腸桿菌的結構數據相比較，推測其基本架構可能和大腸桿菌的ATP合成脢相似；猶如Capaldi提出的模型次單元b1是位在 次單元III 環的外圍。

ATP synthase is an important enzyme for the living organisms by oxidative phosphorylation as the energy source. The driving force for ATP synthesis is an electrochemical gradient of protons(ΔpH) and /or sodium ion(ΔNa+) generated initially by electron transfer complexes across the mitochondrial, chloroplast, or bacterial membrane. In chloroplast system, only proton can be the driving force for ATP synthesis based on recent studies. We discuss about if Na+ ion and Li+ ion being the driving force of H+- ATP synthase by the method of chemiluminescence. In the analysis data, Na+ ion can be the driving force for ATP synthesis, and its initial rate is 3.5 s-1 under our experimental condition. Although the initial rate is slower than 200 s-1 which driven by proton (Δ pH 4.4), it is faster than 0.7 s-1 which driven by Na+ ion of Propionigenium Modestum. In the case of Li+ ion, it can not drive ATP synthesis but it has the localized hydrolysis with water to produce H+. Therefore, the initial rate is 13.2 s-1 for ATP synthesis as given the membrane potential 140 mV. Simultaneously, we discuss about the effect of proton and Na+ ion or Li+ ion on the same side or the opposite side of the proteoliposome. When Na+ ion presents on opposite side of proton, it does not effect on the initial rate of ATP synthesis which driven by proton under the condition of Δ pH 4.0 and Δ pH 3.3. Nevertheless, Li+ ion can occlude the proton channel of CF0 and slow down the initial rate under the condition of Δ pH 3.3 and can not occupy the binding site and remains the similar initial rate under the condition of Δ pH 4.0. In another aspect, when Na+ ion presents on the same side of proton, it competes with proton to occupy the active binding site of CF0 and slows down the initial rate under the condition of Δ pH 4.0. As for Li+ ion, it also can occupy the active binding site and slow down the initial rate under the condition of Δ pH 3.5 but does not affect on initial rate under the condition of Δ pH 4.4. We also study the interaction of CF1 domain (hydrophilic part) and CF0 domain (hydrophobic part) by dynamic light scattering. The major interaction of these two domains is ionic force and no water participation during the process of association to CF0F1 no matter in oxidized from or reduced form of H+-ATP synthase. It is more stable of the oxidized form than the reduced form by comparison of the dissociation constants and thermodynamic constants. Since the importance of ATP synthase, hundreds of papers concerning the structure of ATP synthase using the material of E coli and mitochondria have been published last decade years. The structure is consistent of two parts: one is the hydrophilic part of CF1 domain whose function is the reaction canter of ATP synthesis or hydrolysis ; the other is the hydrophobic part of CF0 domain whose function is applying chemical potential by Na+ ion and/or proton gradient across the membrane. However, there are some special characters of the chloroplast system like latent state / active state, so we study the scaffold structure of H+-ATP synthase from the chloroplast system by the technique of single molecule fluorescence resonance energy transfer. First, we labeled TMR and Cy5 on the subunit b1 and ε, and then incorporate the intact CF0F1 into the liposome. There exist three distinct FRET states and the spatial distances between two subunits were calculated by the Foster theory. They are 5.3 nm, 6.6 nm and 7.6 nm, separately. The ε subunit is located on a circle around the axis of rotation and the b1 subunit is fixed in the stalk which connects the CF0 and CF1. The scaffold structure of H+ -ATP synthase is similar to that of E coli and mitochondria system. The b1 stalk is outside the III ring and the experimental result is agreed with the model of Capaldi suggested.