Abstract

The heliobacterial reaction center (HbRC) is the simplest known photochemical reaction center, in terms of its polypeptide composition. In the heliobacterial cells, its electron donor is a cytochrome (cyt) c₅₅₃ attached to the membrane via a covalent linkage with a diacylglycerol. We have reconstituted purified HbRC into liposomes mimicking the phospholipid composition of heliobacterial membranes. We also incorporated a lipid with a headgroup containing Ni(II):nitrilotriacetate (NTA) to provide a binding site for the soluble version of the heliobacterial cyt c₅₅₃ in which the N-terminal membrane attachment site is replaced by a hexahistidine tag. The HbRC was inserted into the liposomes with the donor side preferentially exposed to the exterior; this bias increased to nearly 100% with higher concentrations (≥ 10 mol%) of the Ni(II)-NTA lipid in the membrane, and is most likely due to the net negative charge of the surface of the membrane. The HbRC in proteoliposomes without the Ni(II)-NTA lipid exhibited normal charge separation and subsequent charge recombination of the P₈₀₀⁺F_X⁻ state in 15 ms; however, the oxidized primary donor (P₈₀₀⁺) was not significantly reduced by added H₆-cyt c₅₅₃. In contrast, with proteoliposomes containing the Ni(II)-NTA lipid, addition of H₆-cyt c₅₅₃ resulted in a new kinetic component resulting from fast reduction (2–5 ms) of P₈₀₀⁺ by H₆-cyt c₅₅₃. The contribution of this kinetic component varied with the concentration of added H₆-cyt c₅₅₃ and could represent 80% or more of the total P₈₀₀⁺ decay. Thus, the HbRC and its interaction with its native electron donor have been reconstituted into an artificial membrane system.

中文翻译：

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重组Heliobacterial光化学反应中心和细胞色素c 553成为脂质体系统

摘要

就其多肽组成而言，Heliobacterial反应中心（HbRC）是最简单的已知光化学反应中心。在Helobacterial细胞中，其电子供体是通过与二酰基甘油的共价键连接到膜的细胞色素（cyt）c ₅₅₃。我们已经将纯化的HbRC重构为脂质体，该脂质体模仿了Heliobacterial膜的磷脂成分。我们还将脂质与头基包含Ni（II）：硝酸三乙酸酯（NTA）的脂质结合在一起，以提供可溶形式的Heliobactery cyt c ₅₅₃的结合位点其中N端膜附着位点被六组氨酸标签代替。将HbRC插入脂质体中，供体侧优先暴露在外部；当膜中的Ni（II）-NTA脂质浓度更高（≥10 mol％）时，这种偏倚会增加到近100％，这很可能是由于膜表面的净负电荷所致。所述HbRC在脂蛋白体，而不在Ni（II）-NTA脂质表现出正常的电荷分离和P的后续电荷重组₈₀₀ ⁺ ˚F _X ^-状态在15毫秒; 但是，添加H ₆ -cyt c ₅₅₃并不能显着还原氧化的主要供体（P ₈₀₀ ⁺）。。相反，对于含有Ni（II）-NTA脂质的蛋白脂质体，H ₆ -cyt c _553的添加导致了新的动力学成分，这是由H ₆ -cyt c ₅₅₃快速还原P ₈₀₀ ⁺（2-5 ms）导致的。。该动力学成分的贡献随添加的H ₆ -cyt c ₅₅₃的浓度而变化，并且可以代表总P ₈₀₀ ⁺衰变的80％或更多。因此，HbRC及其与其天然电子供体的相互作用已被重构为人工膜系统。