Abstract: The site-specific residue replacements E194Q, I119T/T121S/A126T and I119T/T121S /A126T/E194Q were introduced into bop gene and expressed in halobacterium salinarium L₃₃ with the vector pXL-NovR, respectively. The photoreaction of three mutants BR_E194Q, BR_{I119T/T121S/A126T} and BR_{I119T/T121S/A126T/E194Q}(the quadruple mutant) of bacteriorhodopsin(BR) and WT-BR(wild type BR) was investigated in solution and in PVA films at pH=7.0 by UV-Vis absorption spectrum and Resonance Raman spectra. The lifetime of M intermediate of the three mutants in PVA films were recorded by employing photomicrography. In distilled water, the visible absorption maximum of BR_{I119T/T121S/A126T} and BR_{I119T/T121S/A126T/E194Q} mutants were blue shifted notably by 11 and 12 nm respectively and the BR_E194Q's was weakly red shifted in comparison with WT-BR. In PVA film, BR_{I119T/T121S/A126T/E194Q} mutant caused a blue shift of the visible absorption spectrum from 568 to 557 nm. The characteristic bands of Resonance Raman spectra of BR_{I119T/T121S/A126T/E194Q} mutant were observed at 1334cm^-1 of the L intermediate, 1200, 1328, 1530 and 1549 cm^-1 of the N intermediate and 1567 and 1573 cm^-1 of M intermediate in solution. However, the 1334 and 1549 cm^-1 bands of the quadruple mutant disappeared and the intensity of 1187 cm^-1 band decreased in PVA film. The lifetime of M intermediate of BR_E194Q was less than 1.0 s by photomicrography. The lifetime of M intermediates of BR_{I119T/T121S/A126T} and BR_{I119T/T121S/A126T/E194Q} were 3.0 s and 2.0 s, respectively.

Key words: Bacterorhodopisin(BR), Mutant, Resonance Raman spectrum, PVA