Halorhodopsins

Hai dka W A, R Henderson and D Oesterhelt 1995. 3-DimensionaI Structure of Halorhodopsin Angstrom Resolution. Journal of Molecular Biology 2A7 726-738. 

FIGURE 10.23 The folding of halorhodopsin with the transmembrane segments indicated. The only lysine residue in the protein is Lys , to which the retinal chromophore is covalently linked. 

FIGURE 10.24 A helical wheel model of halorhodopsin. The amino acids facing the polar, hydrophilic core of the protein are shown. Of these 60 residues, 36 are conserved between halorhodopsin and bacteriorhodopsin. (Adapted from OesterMt, D., and Tittor, f, 1989. Treads ia Biochemical Scieaces 14 57—61.)... 

FIGURE 10.25 The photocycle of light-adapted halorhodopsin (liR), shown in the presence and absence of chloride. The superscripts indicate the maxima of the difference spectra between IrR and the intermediates. 

Sensory rhodopsin (sR) and proteorhodopsin 4.12.1. Sensory rhodopsin Halobacteria contain a family of four retinal proteins, bR, halorhodopsin (hR), sensory rhodopsin I (sR I), and phobor-hodopsin (pR or sensory rhodopsin II, sR II), which carry two distinct functions through common photochemical reactions. In particular, bR... 

Kolbe, M., Besir, H., Essen, L. and Oesterhelt, D. (2000). Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution, Science, 288, 1390-1396. 

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)... 

Prokaryotic organisms have developed a number of transport mechanisms for the extrusion of sodium ions against a concentration gradient. In Escherichia coli sodium efflux is linked to proton uptake and is independent of internal ATP, i.e. the cell uses a sodium/proton antiporter.70,71 In contrast in Streptococcus faecalis,72 sodium efflux involves an ATP-driven sodium-translocating ATPase, while in Halobacterium halobium the protein halorhodopsin has been postulated to catalyze the coupling of Na+ extrusion to light.73... 

Persike N, Pfeiffer M, Guckenberger R, Radmacher M, Fritz M. Direct observation of different surface structures on high-re-solution images of native halorhodopsin. J. Mol. Biol. 2001 310 773-780. 

Bacteriorhodopsin and halorhodopsin are unique energy transducers characteristic of halobacteria and absent in other living cells. The structural and functional aspects of these retinal proteins will be considered in Chapter 6 of this volume. Here we would like to review only two aspects of the problem, namely, the mechanism of the light-induced transmembrane eharge displacement by bacteriorhodopsin, and the involvement of bacteriorhodopsin in photoreception, mediated by a sensor. 

The scheme in Fig. 2 may also explain the mechanism of Cl pumping by halorhodopsin. Apparently, in this case Arg-200 plays a role similar to that of Asp-96 in bacteriorhodopsin. Some other arginines may also take part in Cl transport. It is remarkable that Asp-96 is absent from halorhodopsin (see Chapter 6 of this volume). 

It should be noted in this context that (i) there are no additional arginines in the intramembranous part of halorhodopsin in comparison with bacteriorhodopsin [30] and (ii) bacteriorhodopsin is competent in Cl binding [31-33] and perhaps even in Cl transfer when [H ] is low and [Cl ] is high [34]. 

The specificity of halorhodopsin for the transported anion is not absolute. It can pump not only Cl but also NO]. However, the rate of transport of Cl is much higher than that of NO3. 

Halorhodopsin has also been found in another halophilic microorganism, Natronobac-terium pharaonis. Here it transports NOj as fast as Cl [31]. 

To some extent this has indeed happened. Results so far suggest that bacteriorhodopsin functions in an unexpectedly simple way while incorporating all of what one would expect from an effective energy-transducing system proton transfer across the internal hydrophobic barrier generates a proton-motive force independent of external proton concentration it is essentially irreversible and resists proton back-pressure yet proceeds with minimal thermal losses. The mechanism of chloride transport in halorhodopsin is not yet as clear, but there is reason to believe there is a far-reaching analogy between this system and bacteriorhodopsin. 

Fig. 1. Sequence alignment for three bacteriorhodopsins and two halorhodopsins. For brevity the single-letter amino acid code is used. Alignment and helical assignments are as in ref. [17]. Designations AR-1, a bacteriorhodopsin from Halobacterium sp. aus-1 [44] AR-2, a bacteriorhodopsin from Halobacterium sp. aus-2 [45] BR, bacteriorhodopsin from H. halobium [5] FIR, halorhodopsin from H. halobium [43] PHR, a halorhodopsin from Natronobacterium pharaonis[ l]. The patterns of dots and asterisks indicate either identity in all five sequences (dots only) or identity among the bacteriorhodopsins and halorhodopsins only (dots and asterisks).

The sequence similarities between bacteriorhodopsins and halorhodopsins from various species [17,43—45] aigue for extensive structural similarities between the two kinds of proteins. It seems likely that the tertiary structure of the halorhodopsins is a generalized bacteriorhodopsin-like arrangement, but two- or three-dimensional crystalline halorhodopsin has not been produced as yet. A crude model for halorhodopsin, based on... 

Fig. 4. Approximate structure of halorhodopsin, based on analogy with bacteriorhodopsin. Putative chloride channels are as indicated with inclined dotted cylinders. arglOS probably participates in chloride uptake arg200 probably participates in chloride release [50],...

Although bacteriorhodopsin contains all of the buried arginine residues which could possibly play a role in binding anions in halorhodopsin, its absorption does not show anion-dependent effects except at very low pH where protonation of asp85 (with a pK of 2.5) causes a shift from 568 nm to about 605 nm [22,56-63]. Addition of chloride to this blue chromophore shifts the maximum back to 565 nm [56,61,64-66]. A sustained photocurrent was not seen at the low pH, but after addition of chloride the photocurrent reappeared. It is tempting to compare these chloride-dependent effects to the behavior of halorhodopsin the possibility of chloride transport by bacteriorhodopsin at low pH was mentioned [67,68]. However, there are discrepancies. Chloride in halorhodopsin causes a red-shift rather than a blue-shift, and the photocycle of bacteriorhodopsin at low pH with bound chloride is quite different from the photocycle of halorhodopsin with bound chloride [61]. 

After a few minutes illumination ( light-adaptation ) bacteriorhodopsin immobilized in the purple membrane lattice contains 100% aW-trans retinal [73-78]. Light-adapted solubilized bacteriorhodopsin [79,80] and halorhodopsin [81-83], on the other hand, contain a mixture of about % aW-trans and /3 n-cii retinal. Dark-adaptation which takes minutes or hours, depending on conditions, results in thermally stable mixtures of /3 a -tmns and % 13-c/s chromophores in all cases. The dark-adapted 13-cis chromophores are stable because the overall shape of retinal is not very different from that of the a -trans chain, the C=N bond having assumed the syn rather than the anti configuration [84,85]. 

The pKa of the protonated Schiff base in bacteriorhodopsin is shifted from around 7 in model compounds in solution to well above 10 [86,87]. In contrast, the halorhodopsin Schiff-base pK is not raised and a pH-dependent equilibrium at 7.4 between the... 

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