Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its a carbon (Ca-H). The labeled membranes containing bR were investigated by difference Fourier transform infrared (FTIR) spectroscopy. It has been derived from K/bR and M/bR difference spectra (K and M are photocycle intermediates) that several bands previously assigned to the retinal chromophore are coupled to the Ca-H. The vibrational modes that exhibit this coupling are principally associated with C15-H and N-H vibrations. [Ca-2H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the Ca-2H label either on lysine-216 and -172 or on the remaining five lysine residues of the protein. FTIR studies of the regenerated bR system, together with methylation of all lysines except the active-site lysine, reveal that the changes observed due to backbone labeling arise from the active-site lysine. The intensity of the C15-H out-of-plane wag is interpreted as a possible indication of a twist around the C15=N bond.Bacteriorhodopsin (bR), a 26-kDa pigment, acts as a lightdriven proton pump in the cell membrane of Halobacterium halobium (1). bR is composed of a chromophore (retinal) covalently linked to an amino acid polypeptide chain through the e-amino group of a lysine residue. The configuration of the chromophore of bR5a (light-adapted, proton-pumping active form) is an all-trans-retinal, and the linkage is through a protonated Schiff base (RSBH+) (2) (Fig. 1). Time-resolved absorption spectra on the light-adapted form reveals a photocycle that is kinetically coupled to proton transport (3,4). In this photocycle, spectroscopically distinct intermediates appear, which are designated J625, K630, L550, M412, N520, and Ow. Resonance Raman (RR) and Fourier transform infrared (FTIR) spectroscopy have been shown to be powerful tools for obtaining structural information on the light-induced structural alterations of the active-site retinal and various amino acids (5, 6). In all of these investigations, the possibility of structural alterations in the active-site lysine has not been addressed. McMaster and Lewis (7) have focused on the problem of lysine, and they showed that numerous bands in the previously investigated FTIR difference spectra associated with light absorption by the chromophore involved lysine. In this paper, we extend these measurements to include contributions to the FTIR spectra of the backbone of lysine. We prove that these contributions arise from the lysine that is complexed to the retinal chromophore, and we show that active-site lysine backbone vibrations are strongly coupled to modes that have been assigned previously to retinal. Furthermore, we observe that the nature of the coupling to the backbone is altered in going from bR to K.MATERIALS AND METHODS Lysine deuterated at the a carbon (Ca-H) was synthesized in a similar fashion to the method of Johns and Whelan (8). Incorporation into bR was carried out by growing halobacteria on a defined medium in which lysine was repl...
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