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Title: The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle

Abstract

During the L {r_arrow} M reaction of the bacteriorhodopsin photocycle the proton of the retinal Schiff base is transferred to the anionic D85. This step, together with the subsequent reprotonation of the Schiff base form D96 in the M {r_arrow} N reaction, results in the translocation of a proton across the membrane. The first of these critical proton transfers occurs in an extended hydrogen-bonded complex containing two negatively charged residues (D85 and D212), two positively charged groups (the Schiff base and R82), and coordinated water. We simplified this region by replacing D212 and R82 with neutral residues, leaving only the proton donor and acceptor as charged groups. The D212N/R82Q mutant shows essentially normal proton transport, but in the photocycle neither of this protein nor of the D212N/R82Q/D96N triple mutant does a deprotonated Schiff base (the M intermediate) accumulate. Instead, the photocycle contains only the K, L, and N intermediates. Infrared difference spectra of D212N/R82Q and D212N/R82Q/D96N demonstrate that although D96 becomes deprotonated in N, D85 remains unprotonated. On the other hand, M is produced at pH>8, where according to independent evidence the L {leftrightarrow} M equilibrium should shift toward M. Likewise, M is restored in the photocycle when the retinalmore » is replaced with the 14-fluoro analogue that lowers the pK{sub a} of the protonated Schiff base, and now D85 becomes protonated as in the wild type. We conclude from these results that the proton transfers to and from the Schiff base, and now D85 becomes protonated as in the wild type. We conclude form these results that the proton transfers to and from the Schiff base probably both occur after photoexcitation of D212N/R82Q, but the L {leftrightarrow} M and M {leftrightarrow} N equilibria are shifted away from M, and, untypically, D85 does not retain the proton it had gained. 72 refs., 11 figs., 1 tab.« less

Authors:
; ;  [1]
  1. Univ. of California, Irvine, CA (United States); and others
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
430188
DOE Contract Number:  
FG03-86ER13525; FG02-92ER20089
Resource Type:
Journal Article
Journal Name:
Biochemistry (Eaton)
Additional Journal Information:
Journal Volume: 34; Journal Issue: 39; Other Information: PBD: 3 Oct 1995
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; BACTERIA; PHOTOCHEMICAL REACTIONS; PROTON TRANSPORT; RHODOPSIN; MUTANTS; ISOMERIZATION; SCHIFF BASES

Citation Formats

Brown, L S, Varo, G, and Lanyi, J K. The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle. United States: N. p., 1995. Web. doi:10.1021/bi00039a053.
Brown, L S, Varo, G, & Lanyi, J K. The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle. United States. doi:10.1021/bi00039a053.
Brown, L S, Varo, G, and Lanyi, J K. Tue . "The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle". United States. doi:10.1021/bi00039a053.
@article{osti_430188,
title = {The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle},
author = {Brown, L S and Varo, G and Lanyi, J K},
abstractNote = {During the L {r_arrow} M reaction of the bacteriorhodopsin photocycle the proton of the retinal Schiff base is transferred to the anionic D85. This step, together with the subsequent reprotonation of the Schiff base form D96 in the M {r_arrow} N reaction, results in the translocation of a proton across the membrane. The first of these critical proton transfers occurs in an extended hydrogen-bonded complex containing two negatively charged residues (D85 and D212), two positively charged groups (the Schiff base and R82), and coordinated water. We simplified this region by replacing D212 and R82 with neutral residues, leaving only the proton donor and acceptor as charged groups. The D212N/R82Q mutant shows essentially normal proton transport, but in the photocycle neither of this protein nor of the D212N/R82Q/D96N triple mutant does a deprotonated Schiff base (the M intermediate) accumulate. Instead, the photocycle contains only the K, L, and N intermediates. Infrared difference spectra of D212N/R82Q and D212N/R82Q/D96N demonstrate that although D96 becomes deprotonated in N, D85 remains unprotonated. On the other hand, M is produced at pH>8, where according to independent evidence the L {leftrightarrow} M equilibrium should shift toward M. Likewise, M is restored in the photocycle when the retinal is replaced with the 14-fluoro analogue that lowers the pK{sub a} of the protonated Schiff base, and now D85 becomes protonated as in the wild type. We conclude from these results that the proton transfers to and from the Schiff base, and now D85 becomes protonated as in the wild type. We conclude form these results that the proton transfers to and from the Schiff base probably both occur after photoexcitation of D212N/R82Q, but the L {leftrightarrow} M and M {leftrightarrow} N equilibria are shifted away from M, and, untypically, D85 does not retain the proton it had gained. 72 refs., 11 figs., 1 tab.},
doi = {10.1021/bi00039a053},
journal = {Biochemistry (Eaton)},
number = 39,
volume = 34,
place = {United States},
year = {1995},
month = {10}
}