Spectroscopic Study of Proton-Transfer Mechanism of Inward Proton-Pump Rhodopsin, Parvularcula oceani Xenorhodopsin
Spectroscopic Study of Proton-Transfer Mechanism of Inward Proton-Pump Rhodopsin, Parvularcula oceani Xenorhodopsin
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DOI：
10.1021/acs.jpcb.8b01279
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发表时间：
2018-06-28
影响因子：
3.3
通讯作者：
Kandori, Hideki
中科院分区：
文献类型：
--
作者：
Inoue, Keiichi;Tahara, Shinya;Kandori, Hideki
海洋小球藻紫质是第一个光驱动的内向质子泵。为了了解质子向内传输的机理，我们进行了全面的瞬时吸收光谱。超快泵浦探测光谱分析表明，视网膜的异构化时间为1.2ps，远低于其他微生物视紫红质的异构化时间(180-770fs)。J产生后，K中间体在4ps时形成。质子转移发生在较慢的时间尺度上。通过监测pH指示染料吡喃的瞬时吸收变化，观察到质子在L/M-to-M和M衰变过程中的释放和吸收。在诱变实验中，虽然Asp216中有一个质子被释放到细胞质中，但在胞外侧没有发现质子供体残基。我们发现，在摄取质子的同时，视网膜发生了分支异构化(从13-顺-15-抗到13-顺-15-syn和全反-15-抗)。此外，尽管质子释放表现出很大的动力学同位素效应，但质子吸收的动力学同位素效应可以忽略不计。这些结果表明，视网膜异构化是质子摄取的限速过程，通过热异构化调节视网膜Schiff碱的PK，使其能够从细胞外介质摄取。这种质子吸收机制不同于具有内部质子供体的向外质子泵的质子吸收机制，对于理解膜蛋白的离子传输方向是重要的。
Parvularcula oceani xenorhodopsin is the first light-driven inward proton pump. To understand the mechanism of inward proton transport, comprehensive transient absorption spectroscopy was conducted. Ultrafast pump-probe spectroscopy revealed that the isomerization time of retinal is 1.2 ps, which is considerably slower than those of other microbial rhodopsins (180-770 fs). Following the production of J, the K intermediate was formed at 4 ps. Proton transfer occurred on a slower timescale. Proton release and uptake were observed on the L/M-to-M and M decay, respectively, by monitoring transient absorption changes of pH-indicating dye, pyranine. Although a proton was released from Asp216 into the cytoplasmic medium, no proton-donating residue was identified on the extracellular side in mutation experiments. We revealed that a branched retinal isomerization (from 13-cis-15-anti to 13-cis-15-syn and all-trans-15-anti) occurred simultaneously with proton uptake. Furthermore, although the proton release showed a large kinetic isotope effect (KIE), the KIE of proton uptake was negligible. These results suggest that retinal isomerization is the rate-limiting process in proton uptake and that the regulation of pK, of the retinal Schiff base by thermal isomerization enables the uptake from extracellular medium. This proton uptake mechanism differs from that of the outward proton pump with an internal proton donor and is important for understanding how the direction of ion transport by membrane proteins is determined.