Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin

Abstract

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface.

Membrane potential stabilizes the O intermediate in liposomes containing bacteriorhodopsin

“…Apparently, the long-lived O intermediate may be formed both from the trans-bR and 13-cis-bR molecules. This contradicts the conclusion of Nasuda-Koyuama et al [12] that only 13-cis-bR is able to give the bathoform in response to low pH in the intravesicular volume.…”

“…Thus, the above data indicate that the membrane potential is the main cause for the flash‐induced formation of the long‐lived intermediate. However, they cannot exclude that its formation under continuous illumination may also be caused by acidification of the intravesicular volume as was suggested by Nasuda‐Koyuama et al [12]. It is noteworthy that light adaptation increases the magnitude of the O intermediate concurrently with the increase in the M intermediate magnitude (data not shown).…”

“…Nasuda‐Koyama et al [12] described formation of a blue form in bR liposomes under continuous illumination. The authors suggested that this effect is due to the formation of the blue acid bR form induced by a large decrease in the intravesicular pH.…”

Structure of archaerhodopsin-2 at 1.8 Å resolution

“…In fact, the lipid component (for example triglycolipid) observed in the P622 crystal is partially removed or missing in other crystal forms. It has been reported that the neutral purple form is destabilized in bR-containing vesicles reconstituted with egg lethicin, in which the transition from the neutral purple form into an inactive red form with max at 480 nm takes place at pH 6.5 (Nasuda- Kouyama et al, 1990).…”

Structural Role of Bacterioruberin in the Trimeric Structure of Archaerhodopsin-2

“…32 In the monomeric state, on the other hand, the neutral purple conformation is stable in a very restricted range of pH; i.e., the acid blue transition and the alkaline red transition are observed at pH ∼ 4 and ∼ 7, respectively, when bR monomers are incorporated into vesicles composed of egg phosphatidyl choline. 43 These observations suggest that trimerization is effective in maximizing the proton pumping power, which is defined by the largest pH gradient that the proton pump can generate across the membrane. For halobacteria living in alkaline lakes, however, generation of a large pH gradient (i.e., alkalization of the cytosol) may be harmful.…”