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TIME‐RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR‐185→E: ASP‐96 REPROTONATES DURING O FORMATION; ASP‐85 AND ASP‐212 DEPROTONATE DURING O DECAY
Author(s) -
Bousché Olaf,
Sonar Sanjay,
Krebs Mark P.,
Khorana H. Gobind,
Rothschild Kenneth J.
Publication year - 1992
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1992.tb09732.x
Subject(s) - bacteriorhodopsin , deprotonation , protonation , chemistry , halobacteriaceae , chromophore , fourier transform infrared spectroscopy , crystallography , schiff base , stereochemistry , infrared spectroscopy , spectroscopy , photochemistry , halobacterium salinarum , membrane , biochemistry , organic chemistry , ion , physics , quantum mechanics
— The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time‐resolved Fourier transform infrared (FTIR) difference spectroscopy and site‐directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem . 266 , 11063–11067, 1991) we found that Asp‐96 undergoes a deprotonation during the M→N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp‐85 and Asp‐212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr‐185→Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time‐resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all‐ trans O chromophore appear in the time‐resolved FTIR difference spectra in the same time range as the appearance of a red‐shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp‐85, Asp‐96, Asp‐115 and Asp‐212, we conclude that during O formation: (i) Asp‐96 undergoes reprotonation. (ii) Asp‐85 may undergo a small change in environment but remains protonated. (iii) Asp‐212 remains partially protonated. In addition, reisomerization of the chromophore during the N→O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.