Devanathan S, Salamon Z, Tollin G, Fitch J, Meyer T E, Cusanovich M A
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Biochemistry. 2004 Dec 28;43(51):16405-15. doi: 10.1021/bi0481904.
The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to 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 ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 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 a K(D) of 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 cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 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 approximately 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.
利用表面等离子体波导共振光谱法，已高精度测定了来自荚膜红细菌的氧化型和还原型野生型细胞色素c(2)以及细胞色素c(2)赖氨酸93到脯氨酸突变体与光合反应中心（球形红细菌）结合的解离常数。对于所报道的研究，将去污剂增溶的光合反应中心交换到磷脂酰胆碱脂质双层中以近似生理环境。在生理相关离子强度（约100 mM）下，我们发现还原型野生型细胞色素有两个结合位点（K(D)=10和150 nM），其亲和力随离子强度降低而降低（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。利用表面等离子体波导共振光谱法分辨质量变化和结构各向异性对细胞色素结合贡献的能力，我们可以证明两个结合位点具有非常不同的性质。相比之下，氧化型野生型细胞色素在高离子强度下仅与一个K(D)为10 nM的位点结合，且该位点具有与还原型细胞色素低亲和力结合位点相似的性质。氧化型细胞色素c(2)的结合具有强烈的离子强度响应，从高离子强度到低离子强度时亲和力降低约30倍。K93P突变体在两种氧化还原状态下均与一个位点结合，就质量和结构各向异性而言，该位点与氧化型野生型位点相似，在高离子强度下突变体氧化态的亲和力比氧化型野生型细胞色素弱约30倍。因此，还原型野生型细胞色素可与高亲和力和低亲和力位点结合，而氧化型野生型细胞色素以及突变体细胞色素的两种氧化还原状态只能与低亲和力位点结合，这可能是还原型野生型细胞色素结构更稳定的结果。总体而言，这些结果与一个模型一致，即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型细胞色素从反应中心 - 细胞色素复合物中解离，从而促进周转。
