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

Abstract

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...

Effects of individual genetic substitutions of arginine residues on the deprotonation and reprotonation kinetics of the Schiff base during the bacteriorhodopsin photocycle

“…The experimental set up for obtaining the transient absorption at 405 and 296 + 5 nm is basically the same as described previously (31,32), except that the data acquisition and analysis were done by a personal computer. The extraction of the rate constant for both the rise and the decay of the M intermediate and the UV transient absorption were done by using a nonlinear least-square fitting computer program assuming biexponential rise or decay, with variable preexponential factors as shown below.…”

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

“…Tables Table 1. Comparison of natural variation and introduced mutations in retinal-binding pocket of GR and homologs. E [53] N [53] V [54] S [54] T [55] F [56] A [57] D [58] N [59] V [60] A [61] V [61] A [62] T [62] V [62] A [26] -C [63] -C [60] A [26] C [57] V [57] A [63] F [56] F [64] A [65] L [66] V [60] F [67] E [68] N [26] T [26] - G [25] A [25] A [25] K [70] ---------H [25] N [25] --E [71] --a Homologous residues in the retinal-binding pocket were identified through structure-guided alignment of protein sequences. b Natural variation represents variants retrieved from BLAST searches of the NCBI database with GR, BR and PR.…”

Structure of a retinal chromophore of dark-adapted middle rhodopsin as studied by solid-state nuclear magnetic resonance spectroscopy

“…It also suggested that Tyr185 in MR can interact with Asp213 or Thr216 via hydrogen bonding, e.g., Tyr174-Thr204 in Np SRII [ 43 ]. The conserved Tyr is an essential residue in the reaction center of microbial rhodopsins given that its mutants have a significant effect on the efficacy of the light-driven proton pump in BR or the light-induced signal transduction in Np SRII [ 11 , 44 ]. Further, given importance of the formation of the Tyr185-Thr215 hydrogen bond in the signal transduce-able BR mutant with respect to the expression of negative phototaxis [ 20 ], the hydrogen bond between Tyr185 and Thr216 in MR plays an essential role in the signal transduce-able mutant of MR [ 16 ].…”