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

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‐ 13 C]Asp which allowed discrimination between Asp and Glu residues. 14,15‐di 13 C‐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.