Bacteriorhodopsin proton transport

Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2000). Helix deformation is coupled to vectorial proton transport i n the photocycle of bacteriorhodopsin. Nature 406, 645-648. 

Takeda, K., Matsui, Y, Sato, H., Hino, T., Kanamori, E., Okumura, T., Yamane, T., Iizuka, T., Kamiya, N., Adachi, S., and Kouyama, T. Sliding of G-helix in bacteriorhodopsin during proton transport. To be published. 

Lanyi JK. A structural view of proton transport in bacteriorhodopsin. In Biophysical and Structural Aspects of Bioenergetics. Wiksttom M, ed. 2005. Royal Society of Chemistry, Cambridge, UK. 

Dissociation of the purple membrane lattice, followed by incorporation of the bacteriorhodopsin into liposomes [327,356,357], yields a functional proton transport system. Thus, bacteriorhodopsin monomers will translocate protons upon illumination. 

Different aspects of bacteriorhodopsin functioning have been studied early [35-37, 223, 233-249]. However, as a rule, the researchers have described the mechanism of proton transport phenomenologically, though the analysis of the results taken from the FT-IR difference spectra yielded detailed descriptions of the proton state in a proton pathway in all of the above-mentioned intermediates (see, e.g., Zundel [6]). 

Below we propose a microscopic model of the light-induced proton transport in bacteriorhodopsin, which is based on the active site model proposed in Refs. 6, 35-37, 233, and 248. Namely, we shall analyze the influence of the charge separation in the excited chromophore on a set of proton absorption bands covering almost all the visible range [221,222]. 

Further evidence for the light-driven proton translocation by bacteriorhodopsin was obtained by Racker and Stoeckenius who carried out reconstitution ofthe purple membrane into phospholipid vesicles. In the light, the reconstituted vesicles took up protons from the exterior at a rate of 50-200 ng per mg of bacteriorhodopsin, and released them in the dark. The rate of proton uptake in the fight and release in the dark was accelerated by the addition ofvalinomycin, while uncouplers of oxidative phosphorylation abolished the uptake of protons altogether. Note that the direction of proton transport from outside to the inside of the vesicle reported by the authors was opposite to that observed in intact cells, the possibility that bacteriorhodopsin might be oppositely oriented in cells and vesicles, was subsequently confirmed by freeze-etch electron microscopy. 

Bacteriorhodopsin is the quintessential transmembrane ion pun ). It consists of a small, seven-helix protein where proton transport across the membrane is driven by photoisomerization of retinal from the all trans to the 13-cis,l5-anti configuration. A number of high-resolution crystal structures of the protein and its photointermediates have been used to propose several competing mechanisms describing proton translocation to fhe extracellular surface. Unresolved issues include understanding how conformational changes couple to proton transfer and the role played by water molecules in the proton transfer process. ... 

Consideration of relative proton affinities alone is not sufficient to explain the directionality of transport in proton pumps. For efficient proton pumping it is essential that the activated proton (state 2) cannot flow back to group A, which thermodynamically would be dictated by the fact that A has much higher proton affinity than C. To that effect, relative insulation to proton transport in state 2 is required either between B and A, or alternatively, between A and surface I. This requirement is often described in terms of an alternating access model [Jardetzky, 1966] and is now fairly well understood in bacteriorhodopsin [Lanyi, 1999]. Likewise, proton leakage from C to B must be prevented in step 4, where B has a much higher proton affinity than C. In the depicted scheme (Fig.2), the back flow of is hampered by the high proton activity of C relative to O, so that proton relay to B from A is rapid compared to alternative reprotonation from O via C. 

The essential role of protein structure dynamics as a mechanistic consideration in proton transport has focused attention on amino acids such as proline (3, 9), which can confer localized flexibility within and between helical segments. Except for the unique case of bacteriorhodopsin, which is amenable to spectroscopic examination (10), little is known about the nature of molecular dynamics in transport enzymes. 

The usual technique to determine the dynamic helix character of the transmembrane segment is CD spectroscopy. In bacteriorhodopsin, for example, the helical content of the protein changes drastically during the light-induced reaction cycle leading to proton transport (Grigorieff et al., 1996). 

Applications of solid state NMR along with FTIR and Raman spectroscopy and X-ray crystallography to study the structural changes in the proton transport cycle of the light-driven pump, bacteriorhodopsin, have been reviewed by Laniy. " ... 

The hght-induced proton translocation by bacteriorhodopsin at the planar interface of octane/water [10,12,19,20] and in octane-water emulsions [64] has been studied. A retinotoxin thought to form a stable shift base with retinol in rhodopsin, inhibited the light-actived proton transport [64]. 

At the molecular level, it is the light-induced trans-to-cis isomerization of the chromophore retinal that drives the vectorial proton transport. A detailed understanding of the molecular events leading to proton transport was greatly enhanced by elucidation of the crystal structures of ground-state bacteriorhodopsin and several intermediates of the photocycle. The detailed picture is gradually emerging but is still in a state of flux. Periodic reviews were provided by Lanyi. - ... 

When Mitchell first described his chemiosmotic hypothesis in 1961, little evidence existed to support it, and it was met with considerable skepticism by the scientific community. Eventually, however, considerable evidence accumulated to support this model. It is now clear that the electron transport chain generates a proton gradient, and careful measurements have shown that ATP is synthesized when a pH gradient is applied to mitochondria that cannot carry out electron transport. Even more relevant is a simple but crucial experiment reported in 1974 by Efraim Racker and Walther Stoeckenius, which provided specific confirmation of the Mitchell hypothesis. In this experiment, the bovine mitochondrial ATP synthasereconstituted in simple lipid vesicles with bac-teriorhodopsin, a light-driven proton pump from Halobaeterium halobium. As shown in Eigure 21.28, upon illumination, bacteriorhodopsin pumped protons... 

Proton gradients can be built up in various ways. A very unusual type is represented by bacteriorhodopsin (1), a light-driven proton pump that various bacteria use to produce energy. As with rhodopsin in the eye, the light-sensitive component used here is covalently bound retinal (see p. 358). In photosynthesis (see p. 130), reduced plastoquinone (QH2) transports protons, as well as electrons, through the membrane (Q cycle, 2). The formation of the proton gradient by the respiratory chain is also coupled to redox processes (see p. 140). In complex III, a Q,cycle is responsible for proton translocation (not shown). In cytochrome c oxidase (complex IV, 3), trans-... 

Halorhodopsiti. In addition to bacteriorhodopsin there are three other retinal-containing proteins in membranes of halobacteria. From mutant strains lacking bacteriorhodopsin the second protein, halorhodopsin, has been isolated. It acts as a light-driven chloride ion pump, transporting Cl from outside to inside. Potassium ions follow, and the pump provides a means for these bacteria to accumulate KC1 to balance the high external osmotic pressure of the environment in which they live.578 The amino acid sequences of halorhodopsins from several species are very similar to those of bacteriorhodopsin as is the three-dimensional structure.589 However, the important proton-carrying residues D85 and D96 of bacteriorhodopsin are replaced by threonine and alanine, respectively, in halorhodopsin.590 Halorhodopsin (hR)... 

Cells drive active transport in a variety of ways. The plasma-membrane Na+-K+ pump of animal cells (a) and the plasma-membrane H+ pump of anaerobic bacteria (b) are driven by the hydrolysis of ATP. Eukaryotic cells couple the uptake of neutral amino acids to the inward flow of Na+ (c). Uptake of /3-gal actosidcs by some bacteria is coupled to inward flow of protons (d). Electron-transfer reactions drive proton extrusion from mitochondria and aerobic bacteria (e). In halophilic bacteria, bacteriorhodopsin uses the energy of sunlight to pump protons (/). E. coli and some other bacteria phosphorylate glucose as it moves into the cell and thus couple the transport to hydrolysis of phosphoenolpyruvate (g). 

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