Participation of bacteriorhodopsin active-site lysine backbone in vibrations associated with retinal photochemistry

Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its o carbon (Cα - 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 Cα - H. The vibrational modes that exhibit this coupling are principally associated with C15 - H and N - H vibrations. [Cα-²H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the Cα - 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.