To elucidate the role of tyrosine residues in the shift of λmax and the light-driven proton pump of bacteriorhodopsin, the photochemical reaction of tyrosine-iodinated bacteriorhodopsin (tyr-mod-bR) was investigated by low-temperature spectrophotometry. After 4–5 of 11 tyrosine residues of bacteriorhodopsin were iodinated, the meta-intermediate of tyr-mod-bR in 75% glycerol solution became so stable that its decay could be observed even at room temperature and it was stable in the dark for several hours at −65°C.
Four batho-intermediates were formed by irradiation with green light (500 nm) at −170°C. Like native bacteriorhodopsin, these batho-intermediates were photoreversible at −170°C. Four corresponding meta-intermediates were also formed by irradiation at −60°C. Using the difference spectra between meta-intermediates and tyr-mod-bR, the absorption spectra of four kinds of tyr-mod-bRs, batho-intermediates, and meta-intermediates were estimated. Each was at shorter wavelengths than that of its corresponding type in native bacteriorhodopsin. The results indicate that two or more tyrosine residues have some role in determining color in native bacteriorhodopsin.