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

A dual beam pump/probe technique has been 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 degrees C. These studies show that bathorhodopsin has a protonated Schiff base vibration at 1657 cm(-1) which shifts upon deuteration to 1625 cm(-1). Within our 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=N bond and the degree of protonation of the Schiff base nitrogen are the same in bathorhodopsin, rhodopsin, and isorhodopsin. The implication 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 is found to have lines at 853, 875, 920, 1006, 1166, 1210, 1278, 1323, 1536, and 1657 cm(-1). Also, by using an intensified vidicon detector, we have observed Raman scattering from bathorhodopsin at room temperature by generating a photostationary steady state with pulsed laser excitation. At room temperature the three characteristic lines of bathorhodopsin are found 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 degrees C is the same as that formed at room temperature.