Spectroscopic Study of Firefly Oxyluciferin in Enzymatic Environment on the Basis of Stability Monitoring
Spectroscopic Study of Firefly Oxyluciferin in Enzymatic Environment on the Basis of Stability Monitoring
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基于稳定性监测的酶环境下萤火虫氧化荧光素的光谱研究
DOI：
10.1021/jp411476p
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Hidefumi Akiyama
中科院分区：
文献类型：
--
作者：
Yu Wang;Yuhei Hayamizu;Hidefumi Akiyama
为了了解酶微环境对萤火虫生物发光中发射体氧基荧光素(OL)性质的影响，我们研究了OL与消耗的反应混合物中形成的酶荧光素酶的络合物的光谱特征。通过对原位吸收光谱的监测，分析了荧光素酶环境下OL的酶促合成及其稳定性。对石楠果胶酶的吸收光谱表明，其主要形式为中性的OL，很可能是烯醇形式，发出蓝色荧光(∼450 nm)。发射绿色荧光的单阴离子OL(∼560 nm)与中性的烯醇-OL表现出弱的pH依赖平衡。消耗的反应混合物中几乎完全没有OL的红色发射形式。荧光和生物发光的绿色和红色发射的峰值波长相似，但峰值强度和光谱形状有很大差异。在OL的吸收光谱和荧光光谱中也发现了上述特征，它与催化不依赖于pH的红色生物发光的Luciola cross iataluciferase H433Y突变株形成了络合物。光激发不能再现化学反应产生的生物发光的激发态。可能的原因是化学激发态是由二氧杂环己酮中间体分解后的类酮过渡态形成的，而光学激发态是通过激发中性的烯醇-醇而产生的。不同的荧光素酶只影响生物发光反应过程中的化学过渡态，不影响反应后的基态或光激发态。
To understand the influence of the enzyme microenvironment on the properties of the emitter oxyluciferin (OL) in firefly bioluminescence, we investigated the spectroscopic characteristics of OL in a complex with the enzyme luciferase formed in a consumed reaction mixture. By monitoring the in situ absorption spectra, we analyzed the enzymatic synthesis and the stability of OL in luciferase environment. The absorption spectra of OL inPhotinus pyralisluciferase showed that the dominant form was neutral OL, probably the enol form, which emitted blue fluorescence (∼450 nm). A monoanionic OL emitting green fluorescence (∼560 nm) exhibited a weak pH-dependent equilibrium with the neutral enol-OL. The red-emitting form of OL was almost completely absent from the consumed reaction mixture. The peak wavelengths of the green and red emissions of the fluorescence and bioluminescence were similar, but the peak intensities, and hence the spectral shapes, differed greatly. The above characteristics were also found in the absorption and fluorescence spectra of OL in a complex with the H433Y mutant ofLuciola cruciataluciferase, which catalyzes pH-independent red bioluminescence. Optical excitation could not reproduce the excited states of bioluminescence that was generated from the chemical reaction. The probable reason is that the chemical excited states formed from a keto-like transition state after decomposition of a dioxetanone intermediate, whereas the optical excited states were generated by exciting the neutral enol-OL. Different luciferases only influenced the chemical transition state during the bioluminescence reaction; they did not influence the ground states or optical excited states after the reaction.