Activity and Structure of H+ -ATP synthase from Chloroplast
Date Issued
2007
Date
2007
Author(s)
Chen, Mei-Fang
DOI
en-US
Abstract
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.
Subjects
H+ -ATP合成脢
離子通道
葉綠體
蛋白質蛋白質間的作用力
動態光散射光譜
單分子螢光共振能量轉移
H+ - ATP synthase
ion channel
chloroplast
protein-protein interaction
dynamic light scattering
single molecule fluorescence resonance energy transfer
Type
thesis
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