Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Abstract Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans -bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum ( λ max ) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ max was located at 610 nm and the extinction coefficient ( ϵ max ) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ max at 596 nm and an ϵ max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ max at 543 nm and an ϵ max 0.66-times that of bacteriorhodopsin.