Resonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage

Abstract

A dual beam pump/probe laser technique was used with a 585 nm probe wavelength to obtain maximal resonance enhancement of the Raman lines of bathorhodopsin in a photostationary steady-state mixture at -160.degree. C. Bathorhodopsin has a protonated Schiff base vibration at 1657 cm-1 which shifts upon deuteration to 1625 cm-1. Within the experimental error ( .+-. 2 cm-1) these frequencies are identical to those observed in rhodopsin and isorhodopsin. These effects show that the strength of the C.dbd.N bond and the degree of protonation of the Schiff base nitrogen are the same in bathorhodopsin, rhodopsin and isorhodopsin. The implications of these results for the structure of the retinal chromophore in bathorhodopsin are discussed. The resonance Raman spectrum of pure bathorhodopsin has been generated by accurately subtracting the residual contributions of rhodopsin and isorhodopsin from spectra of the low temperature photostationary mixture. Bathorhodopsin has lines at 853, 875, 920, 1006, 1166, 1210, 1278, 1323, 1536 and 1657 cm-1. Also, by using an intensified vidicon detector, Raman scattering from bathorhodopsin was observed at room temperature by generating a photostationary steady state with pulsed laser excitation. At room temperature the 3 characteristic lines of bathorhodopsin are at 858, 873 and 920 cm-1. The fact that the frequencies of these bathorhodopsin lines are nearly identical at both temperatures implies that the retinal conformation in bathorhodopsin formed at -160.degree. C is the same as that formed at room temperature.