Maiti Tushar Kanti, Engelhard Martin, Sheves Mordechai
Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
J Mol Biol. 2009 Dec 4;394(3):472-84. doi: 10.1016/j.jmb.2009.09.024. Epub 2009 Sep 18.
Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK(a) of 5.8+/-0.1. It was proposed that this pK(a) is associated with the pK(a) of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (<3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK(a) value of the isomerization process is similar to the pK(a) of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.
来自嗜盐菌（Natronomonas pharaonis）的嗜盐视紫红质（NpHR）是视网膜蛋白家族的一员，作为一种光驱动的氯离子泵，在视网膜发色团吸收光后，氯离子通过膜进行运输。在本研究中，我们考察了两个主要问题：（1）控制视网膜发色团与NpHR视蛋白结合的因素，以及（2）NpHR视蛋白催化视网膜异构体热异构化的能力。我们发现，嗜盐菌视紫红质（NpHR）色素从其脱辅基蛋白和全反式视黄醛的重组过程取决于pH值，该过程的pK(a)为5.8±0.1。有人提出，这个pK(a)与结合视网膜发色团的赖氨酸残基（Lys256）的pK(a)有关。色素形成受氯化钠浓度的调节，在3.7 M NaCl时观察到最大产量。在较低浓度的NaCl（<3 M）中色素产量较低，可能是由于脱辅基膜采取了改变的构象，无法形成色素。出乎意料的是，与细菌视紫红质的脱辅基膜不同，NpHR视蛋白由于这些视网膜异构体热异构化为全反式视黄醛，会产生含11-顺式视黄醛和9-顺式视黄醛的色素。异构化速率取决于pH值，在较高pH值时更快。异构化过程的pK(a)值与这些视黄醛结合过程的pK(a)相似，这表明Lys256也参与了异构化过程。异构化与氯化钠浓度无关。然而，在没有氯化钠的情况下，脱辅基蛋白采取的构象并不阻止视黄醛的异构化，但会阻止与赖氨酸残基形成共价键。对NpHR脱辅基蛋白催化的视黄醛异构化的速率和热力学参数分析得出结论，脱辅基膜通过三线态机制催化异构化。
