Identification of the Proton Acceptor of Schiff Base Deprotonation in Bacteriorhodopsin: A Fourier‐transform‐infrared Study of the Mutant Asp85 → Glu in Its Natural Lipid Environment

Abstract

Abstract— In order to assign the proton acceptor for Schiff base deprotonation in bacteriorhodopsin to a specific Asp residue, the photoreaction of the Asp85 → Glu mutant, as expressed in Halobacterium sp. GRB, was investigated by static low‐temperature and time‐resolved infrared difference spec‐troscopy. Measurements were also performed on the mutant protein labeled with [4‐13C]Asp which allowed discrimination between Asp and Glu residues. 14,15‐di13C‐retinal was incorporated to distinguish amide‐II absorbance changes from changes of the ethylenic mode of the chromophore. In agreement with earlier UV‐VIS measurements, our data show that from both the 540 and 610 nm species present in a pH‐dependent equilibrium, intermediates similar to K and L can be formed. The 14 ms time‐resolved spectrum of the 540 nm species shows that a glutamic acid becomes protonated in the M‐like intermediate, whereas the comparable difference spectrum of the 610 nm species demonstrates that in the initial state a glutamic acid is already protonated. In conjunction with earlier observations of protonation of an Asp residue in wild‐type M, the data provide direct evidence that the proton acceptor in the deprotonation reaction of the Schiff base is Asp85.

Glutamic Acid 204 is the Terminal Proton Release Group at the Extracellular Surface of Bacteriorhodopsin

“…, characteristic of the M state (Braiman et al, 1988;Fahmy et al, 1992). As in many earlier reported spectra under these conditions, there is no obvious negative band that would indicate the deprotonation of Glu…”

Cytoplasmic Shuttling of Protons in Anabaena Sensory Rhodopsin: Implications for Signaling Mechanism

“…8,42 The FTIR spectra of D75E liposomes showed the same pattern of carboxylic acid protonation as in the wild-type ASR, displaying a predominance of the 1725 cm K1 or the 1716 cm K1 bands at the appropriate pH values, even though the 1716 cm K1 protonation band of Asp217 could be observed at pH values lower than those in the wildtype (not shown).…”

Influence of the 9-Methyl Group of the Retinal on the Photocycle of Bacteriorhodopsin Studied by Time-Resolved Rapid-Scan and Static Low-Temperature Fourier Transform Infrared Difference Spectroscopy

“…34, No. 41, 1995 13505 mode at 1760 cm-I, due to protonation of Asp85 (Braiman et al, 1988a;Fahmy et al, 1992), indicates that some M has been formed (compare to Figure 2c). Consequently, the spectrum of 9-H-BR550 taken at 213 K ( Figure IC) contains a mixture of L and M intermediates.…”