Department of Biophysics, Medical School, University of Pécs, Pécs, Hungary; Szentagothai Research Center, University of Pecs, Pécs, Hungary.
Department of Biophysics, Medical School, University of Pécs, Pécs, Hungary.
Biophys J. 2022 Jun 7;121(11):2135-2151. doi: 10.1016/j.bpj.2022.04.031. Epub 2022 Apr 29.
Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant K for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.
血红素在兼性光异养细菌球形红杆菌的信号转导机制中起着至关重要的作用。它与转录调控复合物 AppA/PpsR 相互作用，其中 AppA 和 PpsR 分别作为光合作用基因表达的反阻遏物和阻遏物。然而，这种相互作用的机制仍不完全清楚。在这项研究中，我们结合电子顺磁共振（EPR）光谱和Förster 共振能量转移（FRET）来证明 PpsR 中血红素与一个假定的半胱氨酸残基的连接。我们表明，AppA 中的血红素结合会影响蛋白在黑暗适应状态下的荧光性质，这表明与不存在血红素和光适应状态相比，黄素环境的约束较小。我们进行了超快瞬态吸收测量，以便揭示全长 AppA 及其血红素结合域单独的动态过程中的潜在差异。对 CO 结合动力学的比较表明，在全蛋白中，血红素口袋更为开放，这与在 CO 传感器 RcoM-2 中观察到的情况相似，并且表明 AppA 的蓝光利用黄素（BLUF）和传感血红素（SCHIC）结构域之间存在一种通讯途径。我们还使用荧光各向异性测定法定量地研究了 PpsR 与 puc 启动子的单个 DNA 片段结合的亲和力。我们得出的结论是，PpsR 的寡聚化最初是由两个 DNA 片段之一的结合触发的，并且在血红素结合到 PpsR 时，观察到 DNA 结合的解离常数 K 增加了约 10 倍。我们的研究在分子水平上提供了关于血红素调节作用的重要新见解，这种调节作用调节了 R. sphaeroides 中的复杂转录调控，并支持了通过血红素与 AppA 和 PpsR 的结合以及对氧气等气体的感应的两种血红素信号级联。
