Bamberg E, Tittor J, Oesterhelt D
Max-Planck-Institut für Biophysik, Frankfurt am Main, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):639-43. doi: 10.1073/pnas.90.2.639.
Halorhodopsin from Halobacterium halobium was purified and reconstituted with lipids from purple membranes. The resulting protein-containing membrane sheets were adsorbed to a planar lipid membrane and photoelectric properties were analyzed. Depending on light conditions, halorhodopsin acted either as a light-driven chloride pump or as a proton pump: green light caused chloride transport and additional blue light induced proton pumping. In the living cell, both to these vectorial processes would be directed toward the cytoplasm and, compared to ion transport by bacteriorhodopsin, this is an inversed proton flow. Azide, a catalyst for reversible deprotonation of halorhodopsin, enhanced proton transport, and the deprotonated Schiff base in the 13-cis configuration (H410) was identified as the key intermediate of this alternative catalytic cycle in halorhodopsin. While chloride transport in halorhodopsin is mediated by a one-photon process, proton transport requires the absorption of two photons: one photon for formation of H410 and release of a proton, and one photon for photoisomerization of H410 and re-formation of H578 with concomitant uptake of a proton by the Schiff base.
从嗜盐菌中纯化出盐视紫红质，并将其与来自紫膜的脂质进行重构。将所得含蛋白质的膜片吸附到平面脂质膜上，并分析其光电特性。根据光照条件，盐视紫红质既可以作为光驱动的氯离子泵，也可以作为质子泵：绿光引起氯离子运输，额外的蓝光诱导质子泵浦。在活细胞中，这两个矢量过程都将指向细胞质，与细菌视紫红质的离子运输相比，这是一种反向质子流。叠氮化物是盐视紫红质可逆去质子化的催化剂，可增强质子运输，并且13-顺式构型（H410）的去质子化席夫碱被确定为盐视紫红质中这种替代催化循环的关键中间体。虽然盐视紫红质中的氯离子运输由单光子过程介导，但质子运输需要吸收两个光子：一个光子用于形成H410并释放一个质子，另一个光子用于H410的光异构化和H578的重新形成，同时席夫碱摄取一个质子。
