Deprotonation of tyrosines in bacteriorhodopsin as studied by Fourier transform infrared spectroscopy with deuterium and nitrate labeling.
Fourier transform infrared (FTIR) difference spectra are presented for bacteriorhodopsin (BR) at low temperature. Previous FTIR measurements have identified several tyrosine residues that change their absorption characteristics between light-adapted BR and dark-adapted BR, or between intermediates K and M [Dollinger, G., Eisenstein, L., Lin, S.-L., Nakanishi, K., Odashima, K., & Termini, J. (1986) Methods Enzymol. 127, 649-662]. These changes were explained by protonation/deprotonation of tyrosine moieties and perturbation of the protein environment surrounding tyrosines. A tyrosine deprotonation was observed to occur between intermediates K and M. The present studies confine the deprotonation to being between intermediates L and M and show that no tyrosines undergo changes between the K and the L states. Evidence is presented that none of the tyrosines undergoing changes at low temperature can be assigned to tyrosine-64. The environmental changes of these tyrosines are discussed in relation to the proton pumping mechanism. Their spatial relation to the chromophore is also discussed. At least two tyrosines are suggested to reside close to the retinal binding site. The reactive groups of the nitrated tyrosine-64 are speculated to be remote from the Schiff base and the active tyrosines but can possibly interact sterically with the ionone ring of the retinal.
Department of Physics, University of Illinois, Urbana-Champaign 61801.
S L
SL
Lin
P
P
Ormos
L
L
Eisenstein
R
R
Govindjee
K
K
Konno
K
K
Nakanishi
D001436:Bacteriorhodopsins / Q000378:metabolism; D003903:Deuterium; D005583:Fourier Analysis; D006217:Halobacterium / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives; D013055:Spectrophotometry, Infrared / Q000379:methods; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine
D014443:Tyrosine; D001436:Bacteriorhodopsins; D003903:Deuterium; D013774:Tetranitromethane; D008697:Methane