Effect of pH on the photoreaction cycles of bacteriorhodopsin

Kobayashi, Takayoshi; Ohtani, Hiroyuki; Iwai, J.; Ikegami, Akira; Uchiki, Hisao

doi:10.1016/0014-5793(83)81078-3

Cited by 68 publications

(71 citation statements)

References 12 publications

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“…It is known that the chromophore established at pH 2, BR-605, is photolabile (22,24 with intermediates comparable to K-590 and L-550 of the native chromophore BR-570 (24). For the "L" intermediate a lifetime >700 k s is reported (24). In our RR experiments with a spinning cell it is estimated that less than 20% of photoproducts are formed during the transit of sample across the beam.…”

Section: Resultsmentioning

confidence: 75%

“…Although the BR concentration was as low as 3 X M, the resonantly enhanced scattering from the chromophore was about five times more intense than Raman scattering from the gel alone, whose contribution therefore could be easily subtracted. It is known that the chromophore established at pH 2, BR-605, is photolabile (22,24 with intermediates comparable to K-590 and L-550 of the native chromophore BR-570 (24). For the "L" intermediate a lifetime >700 k s is reported (24).…”

Section: Resultsmentioning

confidence: 99%

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Structure of bacteriorhodopsin in the acidified membrane and at high ionic strength: resonance Raman study

Massig¹,

Stockburger

Alshuth

1985

Can. J. Chem.

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Resonance Raman (RR) spectra of the retinylidene Schiffs base (SB) chromophore of bacteriorhodopsin (BR) were obtained from purple membrane (PM) suspensions of Halobacteriumhalobium in three different equilibrium states: (1) the native state at neutral pH; (2) BR-605, the acidified membrane at pH 2; (3) BR-565(Cl−) or BR-545 (F−), at low pH and high ionic strength. In the native state the retinyl chromophore is stabilized by an ion-pair complex of the protonated SB group with a negative carboxylate counterion (A−) of the protein, giving [Formula: see text]. In the acidic form, BR-605, it is inferred from the RR spectra that the chromophore is immobilized. This is ascribed to a breakdown of the ionic interaction by protonation of A−. In the two (3) forms the RR spectra show that the original structure of the chromophore in the native state is reestablished, which is attributed to the formation of [Formula: see text] complexes with the anions. It thus turns out from RR spectroscopic evidence that the retinyl chromophore in BR is largely governed by the [Formula: see text] ion-pair formation, which is stabilized by surrounding water molecules.

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Section: Resultsmentioning

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Structure of bacteriorhodopsin in the acidified membrane and at high ionic strength: resonance Raman study

Massig¹,

Stockburger

Alshuth

1985

Can. J. Chem.

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“…Their work also suggests that the form of bR present without these cations is similar to the blue membrane present at very low pH (38)(39)(40)(41)(42). The batho form of the latter has recently been found to form in 30 psec and to decay within the same time scale as that for normal bR (43). If so, then the L550-to-M412 part of the cycle might not take place in the absence of metal cations.…”

Section: Resultsmentioning

confidence: 86%

On the protein (tyrosine)-chromophore (protonated Schiff base) coupling in bacteriorhodopsin.

Hanamoto¹,

Dupuis²,

El‐Sayed³

1984

Proc. Natl. Acad. Sci. U.S.A.

145

140

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The kinetics of formation of both the tyrosinate ion (from its absorption at 296 nm) and the deprotonated Schiff base (M412) (from its absorption at 404 nm) are studied simultaneously at different pH values (7-11) and temperatures (5-250C). Two formation rates are observed for M412 in agreement with previous observations. The slow one is dominant under physiological conditions and is found to be slightly faster than that for the tyrosinate formation. This is in disagreement with the proposal that the tyrosinate formation is a prerequisite to the deprotonation of the Schiff base (M412). The ratio of the amplitudes of the fast and slow components is found to be sensitive to pH and, at any pH, it can be used to calculate an amino acid pKa value of 9.6. This is explained by proposing the existence of two sites for the protonated Schiff base within the protein. In one site, the Schiff base is near the neutral form of an amino acid residue with a pK. value of 9.6 (giving rise to the slow component), while in the other, it is near its conjugate base. The formation of the tyrosinate ion as well as the formation of the slow and fast components of M412 all have activation energies that are comparable to H-bond energies. A model is suggested to account for this and the comparable deprotonation rates of tyrosine and the slow component of the protonated Schiff base. It involves the reduction of their pKa by their exposure to a positively charged species.Bacteriorhodopsin (bR) is a protein found in the cell membrane of the halophile Halobacterium halobium that transforms visible light into chemical energy (1-4). It is composed of one molecule of retinal covalently bound to the E-amino group of a lysine residue in the surrounding protein via a protonated Schiff base linkage (5,6). When bR absorbs light, it undergoes the following simplified photochemical cycle:Following the L550 -* M412 transformation, protons are ejected from the cell, thus generating an electrochemical gradient. This proton gradient directly drives some metabolic processes such as ATP synthesis and other endergonic processes (1-3, 7-10).The exact mechanism of this proton pump still remains unclear. However, accumulated evidence indicates that the M412 intermediate plays a key role in the photochemical cycle of bR (1, 11). M412 is unique in the unprotonated nature of its retinal-lysine Schiff base bond, as compared with the protonated structure characterizing the parent pigment and all other intermediates (1,(12)(13)(14)(15)(16). Some studies have inferred that the Schiff base deprotonation is closely associated with the proton pump mechanism (17, 18). Studies of the effect of chemically modified tyrosines on the rate of decay of M412 suggest a coupling between some tyrosine residues and M412 (19)(20)(21)(22).Information relating to the molecular aspect of M412 formation has been obtained by Kalisky et al. (17). Their low temperature photolysis studies showed that at temperatures below -80'C, the yield of M412 was markedly increased by a change in the...

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“…Removal of these cations from purple membrane patches (6,7) or acidification (2,8,9) causes a color transition from purple to blue. The absorption maximum of the retinal chromophore is believed to be primarily regulated by the electrostatic interactions between the charged or polar side chains of amino acids and the PSB, as well as certain carbon atoms along the retinal electronic system (10)(11)(12)(13)(14).…”

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confidence: 99%

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Zhang

El‐Sayed

Bonet

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca2+-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca2+-regenerated bR were examined. The important results are as follows: (t) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (h) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (ii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affmity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR.Bacteriorhodopsin (bR) is the only protein in the purple membrane of Halobacterium halobium (1, 2). It contains a polypeptide chain of 248 amino acid residues (3), as well as a single retinylidene chromophore bound via a protonated Schiff base (PSB) to the E-NH2 group of Lys-216. Upon absorption of a photon, bR undergoes a photocycle (4) during which the all-trans to 13-cis isomerization of the chromophore takes place followed by the deprotonation of the PSB, and protons are pumped across the cell membrane. The resulting electrochemical proton gradient is then used by the bacteria for some metabolic processes (e.g., ATP synthesis) (4, 5).Well-washed purple membrane patches contain 3-4 mol o...

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Effect of pH on the photoreaction cycles of bacteriorhodopsin

Cited by 68 publications

References 12 publications

Structure of bacteriorhodopsin in the acidified membrane and at high ionic strength: resonance Raman study

Structure of bacteriorhodopsin in the acidified membrane and at high ionic strength: resonance Raman study

On the protein (tyrosine)-chromophore (protonated Schiff base) coupling in bacteriorhodopsin.

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

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