Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the schiff base

Sriram Subramaniam, Duncan A. Greenhalgh, H. Gobind Khorana

Research output: Contribution to journalArticle

Abstract

In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 ± 0.1 and the apparent pK for protonation of the His85 side chain to be ∼3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.

Original languageEnglish (US)
Pages (from-to)25730-25733
Number of pages4
JournalJournal of Biological Chemistry
Volume267
Issue number36
StatePublished - Dec 25 1992

Fingerprint

Bacteriorhodopsins
Schiff Bases
Aspartic Acid
Protons
Deprotonation
Time and motion study
Protonation
Titration
Absorption spectra
Light

ASJC Scopus subject areas

  • Biochemistry

Cite this

Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the schiff base. / Subramaniam, Sriram; Greenhalgh, Duncan A.; Khorana, H. Gobind.

In: Journal of Biological Chemistry, Vol. 267, No. 36, 25.12.1992, p. 25730-25733.

Research output: Contribution to journalArticle

Subramaniam, Sriram ; Greenhalgh, Duncan A. ; Khorana, H. Gobind. / Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the schiff base. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 36. pp. 25730-25733.
@article{8899733f3d084ae394d1da7dfcf252f1,
title = "Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the schiff base",
abstract = "In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 ± 0.1 and the apparent pK for protonation of the His85 side chain to be ∼3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.",
author = "Sriram Subramaniam and Greenhalgh, {Duncan A.} and Khorana, {H. Gobind}",
year = "1992",
month = "12",
day = "25",
language = "English (US)",
volume = "267",
pages = "25730--25733",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "36",

}

TY - JOUR

T1 - Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the schiff base

AU - Subramaniam, Sriram

AU - Greenhalgh, Duncan A.

AU - Khorana, H. Gobind

PY - 1992/12/25

Y1 - 1992/12/25

N2 - In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 ± 0.1 and the apparent pK for protonation of the His85 side chain to be ∼3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.

AB - In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 ± 0.1 and the apparent pK for protonation of the His85 side chain to be ∼3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.

UR - http://www.scopus.com/inward/record.url?scp=0027043105&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027043105&partnerID=8YFLogxK

M3 - Article

C2 - 1464589

AN - SCOPUS:0027043105

VL - 267

SP - 25730

EP - 25733

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 36

ER -