LOB, CNRS, INSERM, Ecole Polytechnique, Institut Polytechnique de Paris, 91128, Palaiseau Cedex, France.
Photochem Photobiol Sci. 2021 May;20(5):663-670. doi: 10.1007/s43630-021-00052-8. Epub 2021 May 11.
Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.
电子从相邻芳香族残基转移对黄素荧光的猝灭在黄素蛋白中普遍存在。除了在特定的光活性系统中构成一个功能过程外，该过程的时间分辨光谱特征可更普遍地用作活性部位构象和动态的探针。在来自嗜热栖热菌的细菌 RNA 转化黄素酶 TrmFO 的 C51A 变体中，荧光的寿命非常短（~1 ps），并且已知附近的 Tyr343 是主要猝灭剂，这在此处通过在具有其他潜在猝灭剂 Trp283 和 Trp214 修饰的蛋白质变体中观察到的非常相似的动力学得到了证实。当 Tyr343 被修饰为氧化还原非活性的苯丙氨酸时，在皮秒-纳秒时间尺度上观察到较慢且高度多相的动力学，反映了异质电子供体-受体构型。我们证明了位于潜在功能柔性环上的 Trp214 在该变体中对电子供体猝灭有贡献。与其他核酸转化酶的观察结果形成对比的是，这些动力学在温度上几乎是独立的。这表明（a）电子转移反应几乎没有势垒，（b）尽管 Trp214 假定具有柔韧性，但在至少 2 ns 的时间尺度上，不存在不同构型之间的交换。提出了广泛的分子动力学模拟结果，以根据缓慢交换的蛋白质构象来解释这一意外发现。
