On the molecular mechanisms of the Schiff base deprotonation during the bacteriorhodopsin photocycle

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

Stern

et al. 1990

The retinylidene chromophore mutant (Y185F) of bacteriorhodopsin, in which Tyr-185 is substituted by phenylalanine, is examined and compared with wild-type bacteriorhodopsin expressed in Escherichia coli; both were reinstituted similarly in vesicles. The Y185F mutant shows (at least) two distinct spectra at neutral pH. Upon light absorption, the blue species (which absorbs in the red) behaves as if "dead" i.e., neither its tyrosine nor its protonated Schiff base undergoes deprotonation nor does its tryptophan fluorescence undergo quenching.-This result is unlike either the purple species (which absorbs in the blue) or wild-type bacteriorhodopsin expressed in E. coli. As the pH increases, both the color changes and the protonated Schiff base deprotonation efficiency suggest a blue-to-purple transition of the Y185F mutant near pH 9. If this blue-to-purple transition of Y185F corresponds to the blue-to-purple transition of purplemembrane (native) bacteriorhodopsin (occurring at pH 2.6) and of wild-type bacteriorhodopsin expressed in E. coli (occurring at pH 5), the protein-conformation changes of this transition as well as the protonated Schiff base deprotonation may be controlled not by surface pH alone, but rather by the coupling between surface potential and the general protein internal structure around the active site. The results also suggest that Tyr-185 does not deprotonate during the photocycle in purple-membrane bacteriorhodopsin.The retinylidene protein bacteriorhodopsin (bR), the other photosynthetic system besides chlorophyll pigments, is a protein pigment found in the puiple membrane of Halobacterium halobium (1, 2). Light-adapted bR contains an alltrans-retinal, which is covalently bound via a protonated Schiff base (PSB) linkage to the apoprotein, known as bacterioopsin (bO) (1,3,4). Upon absorption of visible light, bR at neutral pH undergoes a photochemical cycle (5, 6):As a result, bR translocates protons from inside to outside the cell, increasing the proton concentration on the outer surface of the membrane (7). The created proton gradient across the membrane is then used to transform ADP into ATP in the flial step of H. halobium photosynthesis (2,5,8).Elucidation of the mechanism of this light-driven proton pump is of fundamental interest from chemical and biological points of view. In an approach to understanding the mechanism, a variety of site-directed mutagenetic methods now permit alteration of any amino acid residue in bO (9-12).Thus, bO mutants that contain single-amino acid substitutions have been prepared and studied by a number of biochemical and biophysical techniques to test current hypotheses regarding bR structure and function (13)(14)(15)(16)(17)(18)(19)(20)(21)(22).Previously, the proton pumping across the membrane has been postulated to occur as protons are transferred through amino acid side chains (23,24). Among the several models involving specific residues (25), a role for tyrosine in lightdriven proton pumping by bR was suggested. Tyrosine deprotonates on the sam...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Effect of genetic modification of tyrosine-185 on the proton pump and the blue-to-purple transition in bacteriorhodopsin.

Jang

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

Stern

et al. 1990

“…The blue form is incapable offorming the M412 intermediate (15,16) but allows isomerization of the retinal and formation of the K610 and L550…”

Section: -Br568 Msmentioning

confidence: 99%

“…intermediates (15)(16)(17). bR that is 75% delipidated (dLbR) does not change the color from purple to blue by deionization (18) but forms M412 with half the efficiency of bR (19).…”

Section: -Br568 Msmentioning

confidence: 99%

See 1 more Smart Citation

Tryptophan fluorescence quenching as a monitor for the protein conformation changes occurring during the photocycle of bacteriorhodopsin under different perturbations.

Jang

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

1989

The step, leading to a proton-pumping process that increases the proton concentration on the outside surface of the membrane (7). The created proton gradient across the membrane is then used to transform ADP into ATP in the final step of the photosynthesis of bR (8). Thus, the understanding of the mechanism of the PSB deprotonation becomes important to our understanding of the molecular mechanism of solar energy storage in nature.bR normally contains bound Ca2' and Mg2+ (9, 10).Acidification or removal of metal cations from bR produces the purple-to-blue color transition (9-14). [15][16][17]. bR contains 8 tryptophan and 11 tyrosine residues out of a total of 248 amino acid residues in the bR polypeptide chain (23). These aromatic residues absorb at a relatively long wavelength (-280 nm) compared with the other amino acid residues. Optical probes of these amino acid residues have been used to understand the protein structure and the interactions of the protein with the retinal in bR and its photocycle intermediates (24-29). Protein fluorescence of bR and its retinal-free form, bacterioopsin, is characteristic of tryptophan fluorescence (30,31). The excitation of tyrosine may indirectly contribute to the tryptophan emission by energy transfer (32). Most of the tryptophan in bR interacts with the retinal in bR (28,29,31). The variation in the observed lifetimes of the tryptophan fluorescence decay components has been suggested (29) to result from the variation of the energy transfer efficiency between excited tryptophan molecules and the retinal in bR. Energy transfer from-tyrosine and tryptophan to the retinal in bR has a quantum yield of 0.7-0.8 and leads to a photocycle identical with that triggered by the excitation of the visible absorption bands of retinal (27).

The Effect of Different Metal Cation Binding on the Proton Pumping in Bacteriorhodopsin

Israel Journal of Chemistry

Yang

Yoo

et al. 1995

The first section of this paper is a detailed summary of studies made by us and others on metal cations binding to deionized bacteriorhodopsin (dIbR) and its variants. Our studies include the luminescence experiments of Eu 3 + binding to dIbR and potentiometric studies of Ca" binding to dIbR, to deionized bR mutants, to bacterioopsin, and to dIbR with its C-terminus removed. The results suggest the presence of two classes of binding sites, one class has two high-affinity constants, and one has one low-affinity constant. For Ca" binding, there is one metal cation in each of the two high-affinity sites which are coupled to the charged aspartates 85 and 212 (known to be in the retinal cavity) but not coupled to each other. The lowaffinity class can accommodate 0-6 Ca" ions and most of them are bound to the surface. Mg" has a slightly smaller value for its binding constant to the highestaffinity site. Thus, one expects more Ca" than Mg" bound to the two high-affinity sites. In the second section, we summarize our recent study on the effect of metal cation charge density (Ca'", Mg", Eu 3 +, Tb 3 +, H03+, Dy3+) on the kinetics of both Schiff base deprotonation and proton transport to the extracellular surface. For all metal cations, the apparent rate constant of the slow components of the deprotonation process is the same as that for the transport process at 22°C. The temperature studies, however, show this apparent equality to be fortuitous and to result from cancellation of the contribution of the energy and entropy of activation. Thus, while the entropy of activation is positive for the deprotonation process, it is negative for the proton transport process. These kinetic parameters depend weakly on the charge density, but in an opposite sense for the two processes. These results suggest that the deprotonation is not the rate-limiting step for the proton transport process. A possible mechanism is proposed in which a hydrated metal cation is used to induce the deprotonation of the protonated Schiff base and to dissociate one of its HzO molecules to donate the proton in the L~M process.b~-:>(bR)*~I460~J625~~IO~L550~M4l2~N~b- H+ +H+In the L sso~M412 step, a proton is transferred from