Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy.
Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy.
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氧化和还原细胞色素 c2 与光合反应中心的结合：等离子波导共振光谱。
DOI：
10.1021/bi0481904
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Cusanovich,MA
中科院分区：
文献类型：
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作者：
Devanathan,S;Salamon,Z;Tollin,G;Fitch,J;Meyer,TE;Cusanovich,MA
利用等离子体波导共振光谱技术，测定了荚膜红细菌（Rhodobacter capsulatus）的氧化型和还原型细胞色素2（cytochromec 2）与光合反应中心（Rhodobacter sphaeroides）结合的解离常数。对于报道的研究，洗涤剂溶解的光合反应中心被交换成磷脂酰胆碱脂质双层以接近生理环境。在生理相关的离子强度（约100 mM）下，我们发现了还原的野生型细胞色素的两个结合位点（KD= 10和150 nM），亲和力随离子强度的降低而降低（2 - 5倍）。这些结果表明非极性相互作用是决定解离常数的重要因素。利用等离子体激元波导共振光谱法来解析质量变化和结构各向异性对细胞色素结合的贡献的能力，我们可以证明两个结合位点的非常不同的性质。相比之下，氧化的野生型细胞色素在高离子强度下仅结合到具有10 nM的aKD的单个位点，并且该位点具有与用于结合还原的细胞色素的低亲和力位点相似的性质。氧化细胞色素2的结合具有强烈的离子强度响应，从高到低离子强度，亲和力降低约30倍。K93 P突变体结合到一个单一的网站在两个氧化还原状态，这是类似的，在质量和结构各向异性方面，氧化的野生型网站，与突变体氧化态的亲和力是1.30倍弱于氧化的野生型细胞色素在高离子强度。因此，还原的野生型细胞色素可以结合到高亲和力和低亲和力位点，而氧化的野生型细胞色素和突变细胞色素的两种氧化还原状态只能结合到低亲和力位点，这可能是还原的野生型细胞色素的更稳定结构的结果。总的来说，这些结果与细胞色素三维结构中88 - 102区域（所谓的铰链区）的瞬时构象变化驱动氧化细胞色素从反应中心-细胞色素复合物中解离的模型一致，促进了周转。
The dissociation constants for the binding of oxidized and reduced wild-type cytochromec2fromRhodobacter capsulatusand the lysine 93 to proline mutant of cytochromec2to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths (∼100 mM), we found two binding sites for the reduced wild-type cytochrome (KD= 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2−5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with aKDof 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochromec2has a strong ionic strength response, with the affinity decreasing ∼30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being ∼30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88−102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center−cytochrome complex, facilitating turnover.