One Protein, Two Chromophores: Comparative Spectroscopic Characterization of 6,7-Dimethyl-8-ribityllumazine and Riboflavin Bound to Lumazine Protein
One Protein, Two Chromophores: Comparative Spectroscopic Characterization of 6,7-Dimethyl-8-ribityllumazine and Riboflavin Bound to Lumazine Protein
复制标题
DOI：
10.1021/jp507618f
复制
发表时间：
2014-11-20
影响因子：
3.3
通讯作者：
Schleicher, Erik
中科院分区：
文献类型：
--
作者：
Paulus, Bernd;Illarionov, Boris;Schleicher, Erik
我们研究了雷氏发光杆菌的鲁马津蛋白与其生物活性辅因子6，7-二甲基-8-核糖基鲁马津在不同氧化还原状态下的络合物，并与含有核黄素辅因子的样品进行了比较。利用厌氧光还原，我们能够在相似的蛋白质环境中记录这两个辅因子的光吸收动力学。可以证明，该蛋白能够稳定中性的利博卢嗪自由基，产率接近35%。用W-波段连续波EPR和X-波段脉冲Endor光谱进一步研究了利波卢嗪的自由基态。在这里，可以得到g张量的主值和质子超精细耦合(HFC)的几乎完全的映射。值得注意的是，g张量的主成分与各自含有核黄素的蛋白质的主成分相似；然而，质子HFC显示出显著的差异。通过比较含有利博卢嗪的样品的时间分辨光学吸收和荧光数据，可以识别出仅有荧光，但没有任何中间基团或三重态的迹象。这与含有核黄素辅因子的鲁马津蛋白质样品形成了鲜明的对比，对于这种样品，可以使用时间分辨光谱检测到高产率的光生三重态和一些激发的黄素自由基。这些结果清楚地表明，利博卢马嗪是一个氧化还原活性分子，原则上可以作为其他酶反应的辅因子。
We investigated the lumazine protein from Photobacterium leiognathi in complex with its biologically active cofactor, 6,7-dimethyl-8-ribityllumazine, at different redox states and compared the results with samples containing a riboflavin cofactor. Using anaerobic photoreduction, we were able to record optical absorption kinetics from both cofactors in similar protein environments. It could be demonstrated that the protein is able to stabilize a neutral ribolumazine radical with similar to 35% yield. The ribolumazine radical state was further investigated by W-band continuous-wave EPR and X-band pulsed ENDOR spectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings (hfcs) could be obtained. Remarkably, the g-tensors principal components are similar to those of the respective riboflavin-containing protein; however, the proton hfcs show noticeable differences. Comparing time-resolved optical absorption and fluorescence data from ribolumazine-containing samples, solely fluorescence but no signs of any intermediate radical or a triplet state could be identified. This is in contrast to lumazine protein samples containing the riboflavin cofactor, for which a high yield of the photogenerated triplet state and some excited flavin radical could be detected using time-resolved spectroscopy. These results clearly demonstrate that ribolumazine is a redox-active molecule and could, in principle, be employed as a cofactor in other enzymatic reactions.