Magerl Kathrin, Stambolic Ivan, Dick Bernhard
Institute of Physical and Theoretical Chemistry, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany.
Phys Chem Chem Phys. 2017 May 3;19(17):10808-10819. doi: 10.1039/c6cp08370f.
LOV (light-, oxygen- or voltage-sensitive) domains act as photosensory units of many prokaryotic and eukaryotic proteins. Upon blue light excitation they undergo a photocycle via the excited triplet state of their flavin chromophore yielding the flavin-cysteinyl adduct. Adduct formation is highly conserved among all LOV domains and constitutes the primary step of LOV domain signaling. But recently, it has been shown that signal propagation can also be triggered by flavin photoreduction to the neutral semiquinone offering new prospects for protein engineering. This, however, requires mutation of the photo-active Cys. Here, we report on LOV1 mutants of C. reinhardtii phototropin in which adduct formation is suppressed although the photo-active Cys is present. Introduction of a Tyr into the LOV core induces a proton coupled electron transfer towards the flavin chromophore. Flavin radical species are formed via either the excited flavin singlet or triplet state depending on the geometry of donor and acceptor. This photoreductive pathway resembles the photoreaction observed in other blue light photoreceptors, e.g. blue-light sensors using flavin adenine dinucleotide (BLUF) domains or cryptochromes. The ability to tune the photoreactivity of the flavin chromophore inside the LOV core has implications for the mechanism of adduct formation in the wild type and may be of use for protein engineering.
光、氧或电压敏感（LOV）结构域作为许多原核和真核蛋白质的光感受单元。在蓝光激发下，它们通过黄素发色团的激发三重态经历光循环，产生黄素 - 半胱氨酰加合物。加合物的形成在所有LOV结构域中高度保守，并且是LOV结构域信号传导的主要步骤。但最近的研究表明，黄素光还原为中性半醌也可以触发信号传播，这为蛋白质工程提供了新的前景。然而，这需要对光活性半胱氨酸进行突变。在这里，我们报道了莱茵衣藻向光蛋白的LOV1突变体，其中尽管存在光活性半胱氨酸，但加合物的形成受到抑制。在LOV核心中引入酪氨酸会诱导质子耦合电子向黄素发色团转移。根据供体和受体的几何结构，黄素自由基物种通过激发的黄素单重态或三重态形成。这种光还原途径类似于在其他蓝光光感受器中观察到的光反应，例如使用黄素腺嘌呤二核苷酸（BLUF）结构域或隐花色素的蓝光传感器。调节LOV核心内黄素发色团光反应性的能力对野生型中加合物形成的机制有影响，并且可能用于蛋白质工程。
