Abstract
The structural alterationswhich occur in bacterio- rhodopsin(bR)during dark adaptation(BRs70+BRs4a) and the primary phototransition of the dark photocycle (BR648+&D10) have been investigated by Fourier transform infrared andUV difference spectroscopy. Possible contributionsof tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ringper-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exist in BR670 at physiological temperature and thatit protonates during formation ofBR648. A tyrosinate protonation has also been observed at low temperature during the primary phototransitioofnBR570to the red-shifted photoproduct Kea0 (1).However, we now find that no tyrosine protonation change occurs during the primary phototransition ofBR6,, to the red-shifted intermediate Kglo. Through analysis of bR containing isotopically labeled retinals, it was alsodetermined that the chromophore of GDle0xits in a 13-trans,15- cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570,BR548, and &30, it appears that only the 13- trans,l&trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with theSchiff base positive charge in thBR570 chromophore. Isomerization of the chromophore about either the c13=c14or C=N bond disrupts this interaction causinga protonation of the tyrosinate.