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 spectroscopy. Measurements were also performed on the mutant protein labeled with [4-C-13]Asp which allowed discrimination between Asp and Glu residues. 14,15-diC-13-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.