Open AccessResonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage
Author(s) -
Gregory Eyring,
Richard A. Mathies
Publication year - 1979
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.76.1.33
Subject(s) - rhodopsin , resonance (particle physics) , raman spectroscopy , protonation , schiff base , chemistry , resonance raman spectroscopy , raman scattering , chromophore , photostationary state , photochemistry , analytical chemistry (journal) , optics , crystallography , retinal , isomerization , atomic physics , photoisomerization , organic chemistry , ion , biochemistry , physics , catalysis
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°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°C is the same as that formed at room temperature.