PASTEUR, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France.
Phys Chem Chem Phys. 2019 Apr 24;21(17):8743-8756. doi: 10.1039/c8cp06072j.
Flavoproteins often stabilize their flavin coenzyme by stacking interactions involving the isoalloxazine moiety of the flavin and an aromatic residue from the apoprotein. The bacterial FAD and folate-dependent tRNA methyltransferase TrmFO has the unique property of stabilizing its FAD coenzyme by an unusual H-bond-assisted π-π stacking interaction, involving a conserved tyrosine (Y346 in Bacillus subtilis TrmFO, BsTrmFO), the isoalloxazine of FAD and the backbone of a catalytic cysteine (C53). Here, the interaction between FAD and Y346 has been investigated by measuring the photoinduced flavin dynamics of BsTrmFO in the wild-type (WT) protein, C53A and several Y346 mutants by ultrafast transient absorption spectroscopy. In C53A, the excited FAD very rapidly (0.43 ps) abstracts an electron from Y346, yielding the FAD˙-/Y346OH˙+ radical pair, while relaxation of the local environment (1.3 ps) of the excited flavin produces a slight Stokes shift of its stimulated emission band. The radical pair then decays via charge recombination, mostly in 3-4 ps, without any deprotonation of the Y346OH˙+ radical. Presumably, the H-bond between Y346 and the amide group of C53 increases the pKa of Y346OH˙+ and slows down its deprotonation. The dynamics of WT BsTrmFO shows additional slow decay components (43 and 700 ps), absent in the C53A mutant, assigned to excited FADox populations not undergoing fast photoreduction. Their presence is likely due to a more flexible structure of the WT protein, favored by the presence of C53. Interestingly, mutations of Y346 canceling its electron donating character lead to multiple slower quenching channels in the ps-ns regime. These channels are proposed to be due to electron abstraction either (i) from the adenine moiety of FAD, a distribution of the isoalloxazine-adenine distance in the absence of Y346 explaining the multiexponential decay, or (ii) from the W286 residue, possibly accounting for one of the decays. This work supports the idea that H-bond-assisted π-π stacking controls TrmFO's active site dynamics, required for competent orientation of the reactive centers during catalysis.
黄素蛋白通常通过涉及黄素的异咯嗪部分和蛋白质的芳香残基的堆积相互作用来稳定黄素辅酶。细菌 FAD 和叶酸依赖的 tRNA 甲基转移酶 TrmFO 具有通过不寻常的氢键辅助π-π堆积相互作用稳定其 FAD 辅酶的独特性质，涉及保守的酪氨酸（枯草芽孢杆菌 TrmFO 中的 Y346，BsTrmFO）、FAD 的异咯嗪和催化半胱氨酸（C53）的骨架。在这里，通过超快瞬态吸收光谱测量野生型（WT）蛋白、C53A 和几种 Y346 突变体中 BsTrmFO 的光诱导黄素动力学，研究了 FAD 和 Y346 之间的相互作用。在 C53A 中，激发的 FAD 非常迅速（0.43 ps）从 Y346 中提取电子，生成 FAD˙-/Y346OH˙+自由基对，而激发黄素的局部环境（1.3 ps）的弛豫产生其受激发射带的轻微斯托克斯位移。自由基对然后通过电荷重组衰减，主要在 3-4 ps 内，没有 Y346OH˙+自由基的去质子化。据推测，Y346 和 C53 的酰胺基团之间的氢键增加了 Y346OH˙+的 pKa 值并减缓了其去质子化。WT BsTrmFO 的动力学显示出额外的缓慢衰减成分（43 和 700 ps），在 C53A 突变体中不存在，归因于不经历快速光还原的激发 FADox 群体。它们的存在可能是由于 WT 蛋白的结构更具弹性，这得益于 C53 的存在。有趣的是，消除 Y346 供电子特性的突变导致 ps-ns 范围内的多个较慢猝灭通道。这些通道据推测是由于（i）从 FAD 的腺嘌呤部分提取电子，在不存在 Y346 的情况下异咯嗪-腺嘌呤距离的分布解释了多指数衰减，或（ii）从 W286 残基提取电子，可能解释其中一个衰减。这项工作支持了氢键辅助π-π堆积控制 TrmFO 活性位点动力学的观点，这对于催化过程中反应中心的适当取向是必需的。
