Flavin and redox-active disulfide domains of ferredoxin-dependent flavin thioredoxin reductase (FFTR) homodimers should pivot between flavin-oxidizing (FO) and flavin-reducing (FR) conformations during catalysis, but only FR conformations have been detected by X-ray diffraction and scattering techniques. Atomic force microscopy (AFM) is a single-molecule technique that allows the observation of individual biomolecules with sub-nm resolution in near-native conditions in real-time, providing sampling of molecular properties distributions and identification of existing subpopulations. Here, we show that AFM is suitable to evaluate FR and FO conformations. In agreement with imaging under oxidizing condition, only FR conformations are observed for Gloeobacter violaceus FFTR (GvFFTR) and isoform 2 of Clostridium acetobutylicum FFTR (CaFFTR2). Nonetheless, different relative dispositions of the redox-active disulfide and FAD-binding domains are detected for FR homodimers, indicating a dynamic disposition of disulfide domains regarding the central protein core in solution. This study also shows that AFM can detect morphological changes upon the interaction of FFTRs with their protein partners. In conclusion, this study paves way for using AFM to provide complementary insight into the FFTR catalytic cycle at pseudo-physiological conditions. However, future approaches for imaging of FO conformations will require technical developments with the capability of maintaining the FAD-reduced state within the protein during AFM scanning.

中文翻译：

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原子力显微镜引发铁氧还蛋白依赖性黄素硫氧还蛋白还原酶的构象转变

铁氧还蛋白依赖性黄素硫氧还蛋白还原酶 (FFTR) 同二聚体的黄素和氧化还原活性二硫化物结构域在催化过程中应在黄素氧化 (FO) 和黄素还原 (FR) 构象之间旋转，但 X 射线衍射仅检测到 FR 构象和散射技术。原子力显微镜 (AFM) 是一种单分子技术，可以在近自然条件下实时观察具有亚纳米分辨率的单个生物分子，提供分子特性分布的采样和现有亚群的识别。在这里，我们表明 AFM 适用于评估 FR 和 FO 构象。与氧化条件下的成像一致，仅观察到紫色 Gloeobacter violaceus FFTR (GvFFTR) 和同种型 2 的FR 构象丙酮丁醇梭菌FFTR (CaFFTR2)。尽管如此，对于 FR 同源二聚体，检测到氧化还原活性二硫键和 FAD 结合域的不同相对处置，表明关于溶液中中心蛋白核心的二硫键域的动态处置。这项研究还表明，AFM 可以检测 FFTR 与其蛋白质伙伴相互作用后的形态变化。总之，这项研究为使用 AFM 在伪生理条件下提供对 FFTR 催化循环的补充洞察铺平了道路。然而，未来的 FO 构象成像方法需要技术发展，才能在 AFM 扫描过程中保持蛋白质内的 FAD 减少状态。