Aharoni Amir, Ottolenghi Michael, Sheves Mordechai
Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Biophys J. 2002 May;82(5):2617-26. doi: 10.1016/S0006-3495(02)75603-8.
Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.
细菌视紫红质（bR）的特征是存在一个视网膜 - 蛋白质质子化席夫碱共价键，该键对光吸收具有稳定性。我们揭示了在一种13 - 顺式锁定的bR色素（bR5.13；最大吸收波长λ(max)=550 nm）中存在光诱导的质子化席夫碱水解反应，其中关键的C13 == C14双键周围的异构化被刚性环结构所阻止。光水解反应在多烯链上的任何双键周围都不发生异构化的情况下进行，这表明可能是由于视网膜发色团的光诱导极化导致了蛋白质构象的改变。在水解反应过程中形成了两种光中间体，H450（最大吸收波长λ(max)=450 nm）和H430（最大吸收波长λ(max)=430 nm），通过高效液相色谱分析表明它们具有13 - 顺式构型。水解反应后用蓝光照射，这些中间体重新结合到脱辅基膜上以重新形成bR5.13。照射H450中间体形成原始色素，而在中性pH下照射H430会产生一个红移物种（P580），它会热衰减回bR5.13。电子顺磁共振（EPR）光谱表明bR5.13的细胞质侧类似于天然bR的N光中间体的构象。此外，使用具有渗透活性的溶质，我们观察到水解速率取决于细胞质侧的水活性。最后，我们认为水解反应通过结合过程的反向途径进行，并允许捕获一种在结合过程中未积累的新中间体。
