Determination of retinal Schiff base configuration in bacteriorhodopsin

Abstract

Resonance Raman spectra of the BR568, BR54, K625, and L5so intermediates of the bacteriorhodopsin photocycle have been obtained in 1H20 and 2H20 by using native purple membrane as well as purple membrane regenerated with 14,15-13C2 and 12,14-2H2 isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C14-C15 stretch to the normal modes in the 1100-to 1400-cm-' fingerprint region and to characterize the coupling of the C14-C15 stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C=N cis configuration the C14-C15 stretch and the NH rock are strongly coupled, resulting in a large (-50-cm'1) [14,and [12, and L550 spectra to N-deuteration argues that these intermediates have a C=N trans configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR5" -K625) involves isomerization about the ClY=Cl4 bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.Bacteriorhodopsin functions as a photochemical proton pump in the purple membrane of Halobacterium halobium (1). Absorption of light by bacteriorhodopsin's retinal prosthetic group converts the light-adapted pigment, BR568, to the red-absorbing intermediate K625, which thermally decays back to BR568 through the intermediates L550, M412, and 0Ow(2). The initial photochemical step involves a trans --cis isomerization about the C13=C14 bond of retinal (3-6), which is followed by deprotonation of the Schiff base nitrogen in the L550 --M412 transition (7). Recently it has been shown that reprotonation of the Schiff base and thermal reisomerization of the C13=C14 bond occur in the conversion of M412 to O64o (8). In the dark, BR568 converts to darkadapted bactenorhodopsin, which contains a 60:40 mixture of 13-cis and all-trans protonated Schiff base chromophores denoted BR548 and BR568, respectively. It is generally accepted that chromophore isomerization and Schiff base protonation/deprotonation play an active role in the mechanism of this proton pump. An important element in establish-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.ing the orientation and molecular motion of the Schiff base proton in bacteriorhodopsin's photocycle is the configuration of the retinal-lysine Schiff base bond. However, no experimental determination of the C=N configuration has yet been made.Resonance Raman spectroscopy can be used to examine the structure of the protein-bound retinal chromophore in bacteriorhodopsin (9). To interpret these spectra, it is necessary to assign the vibrational lines to specific normal modes and to establish how the vibrations are affected by changes in chromophore geometry. Selective isotopic substitution of the retinal chromophore facilitates the vibrational assignments, while model compounds and normal mode calculations ca...

The synthesis of 13C-labelled retinals

“…These results could be confirmed by Resonance Raman studies (Ref. 30). With this technique the structures of the chromophores in the intermediates could also be established.…”

Synthesis of 8-, 9-, 12-, and 13-mono-13C-retinal

“…These spectra gave the first in situ "C nmr information of the chromophore within a protein, establishing that in dark-adapted bacteriorhodopsin the chromophore occurs in a 40% all-trans protonated Schiff base and 60% 13-cis, 15-cis protonated Schiff base structure which are not interconverting on the "C nmr time scale. These results were strongly supported by resonance Raman (RR) (7,8) and Fourier transform infrared difference (ITIR diff.) (9) vibrational studies of the "C-labelled bacteriorhodopsins and further detailed inforination concerning the chromophore structure was obtained.…”

Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli

“…H-D exchange reduces the intensity of the 1184 cm À1 band and a frequency up-shift occurs (data not shown). The sensitivity of this band towards deuteration of the Schiff base nitrogen argues for the C‚N-H bond in syn-configuration in the 13-cis isomer [28] which is characteristic for the thermally isomerized 13-cis state. Finally, the strong band at 1009 cm À1 can be assigned to the in-plane rocking mode of the two methyl groups of the retinal chain and the weak bands at 882 and 826 cm À1 are due to HOOP (hydrogen-outof-plane) modes [26].…”