Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biochemistry. 2010 Mar 23;49(11):2398-408. doi: 10.1021/bi901483f.
The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using site-specific spin labeling electron paramagnetic resonance (EPR) spectroscopic techniques. The binding was monitored from the perspective of a reporter spin label attached to either the native C34(C) or the engineered C75(C) residue of wild-type PsaC (PsaC(WT)). Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the P(700)-F(X)/PsaC complex, and the P(700)-F(X)/PsaC/PsaD complex. The changes in the EPR spectral line shape and the rotational correlation time of the spin label when PsaC(WT) binds to the PS I core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P(700)-F(X)/PsaC(WT-C34) complex induces further EPR spectral changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PS I core. The binding of several PsaC variants, each lacking one or more key binding contacts with the PsaA/PsaB heterodimer, was monitored using a reporter spin label at C34(C). Our results indicate that the absence of the PsaC-PsaA/PsaB binding contacts causes PsaC to bind in an altered configuration on the PS I core. In particular, the removal of the entire C-terminus (PsaC(C-term)) causes PsaC to dock in a significantly different orientation when compared to the wild-type protein, as indicated by the EPR spectrum of the P(700)-F(X)/PsaC(C-term-C34) complex. Because the PsaC(C-term) variant retains only the symmetric network of PsaC-PsaA/PsaB ionic contacts, the altered EPR spectrum could, in principle, reflect a fraction of reaction centers that contain PsaC bound in the 180 degrees-rotated, C(2)-symmetry-related configuration. The results of this study are used to provide a comprehensive, stepwise mechanism for the binding of PsaC on the PS I core.
使用基于位置的定点自旋标记电子顺磁共振（EPR）光谱技术研究了 PS I 复合物中 PsaC 亚基的组装。通过附着在天然 C34(C)或工程化的 C75(C)残基上的报告自旋标记监测结合情况，该标记附着在野生型 PsaC（PsaC(WT)）上。分析了 PsaC 组装的三个不同阶段：未结合的 PsaC、P(700)-F(X)/PsaC 复合物和 P(700)-F(X)/PsaC/PsaD 复合物。当 PsaC(WT)与 PS I 核心结合时，EPR 光谱线形状和自旋标记的旋转相关时间的变化与组装过程中预期发生的构象变化一致。向 P(700)-F(X)/PsaC(WT-C34)复合物中添加 PsaD 亚基会引起进一步的 EPR 光谱变化，这表明 PsaD 的存在会影响 PsaC 亚基在 PS I 核心上的取向。使用 C34(C)处的报告自旋标记监测了几种 PsaC 变体的结合情况，每种变体都缺少一个或多个与 PsaA/PsaB 异二聚体的关键结合接触。我们的结果表明，缺少 PsaC 与 PsaA/PsaB 结合接触会导致 PsaC 以改变的构象结合到 PS I 核心上。特别是，与野生型蛋白相比，完全去除 C 端（PsaC(C-term)）会导致 PsaC 以明显不同的方向停靠，这一点可以从 P(700)-F(X)/PsaC(C-term-C34)复合物的 EPR 光谱中看出。由于 PsaC(C-term)变体仅保留了 PsaC-PsaA/PsaB 离子接触的对称网络，因此，改变的 EPR 光谱原则上可以反映包含以 180 度旋转、C(2)对称相关构型结合的 PsaC 的一部分反应中心。本研究的结果用于提供 PS I 核心上 PsaC 结合的综合分步机制。
