Photoreaction of bacteriorhodopsin at high pH: origins of the slow decay component of M (original) (raw)

The absorption spectrum of light-adapted purple membrane in 3 M KC1 is dependent on temperature even in the room temperature region. Temperature-induced difference spectra at various pH values suggested that the trans isomer of bacteriorhodopsin, bR570, is in thermal and/or photodynamic equilibrium with several different conformers. The major second conformer occurring at neutral pH had the same spectroscopic properties as the 1 3 4 s isomer, and its content at 35 O C was estimated to be more than 20%. Heterogeneity in the protein conformation became more significant above pH 8, where temperatureinduced difference spectra exhibited a negative peak at 580 nm and a positive peak at 296 nm. This absorption change is very similar to that observed upon the formation of the N intermediate, suggesting that an N-like conformer occurs at high pH and temperature. A significant temperature dependence was also seen in the M decay kinetics at high pH, which were described by two decay components; i.e., the fast decaying M (M? was predominant at low temperature, but the amplitude of the slow component (MS) increased with increasing temperature. It is suggested that Ms is generated upon excitation of the N-like conformer, in which the residue (Asp-96) usually acting as a proton donor to the Schiff base is deprotonated. The N-like conformer could be N itself, because MS was enhanced when N was accumulated by background light. A strong correlation between the amplitude of Ms and the concentration of N was also revealed by the accumulation kinetics of Mf, Ms, and N after the onset of continuous actinic light. This correlation is explained readily by the photoreaction of N leading to Ms. These results do not necessarily exclude other origins of Ms, however. To explain a large amplitude of Ms at extremely high pH (pH > lo), we may have to assume another ground state in which Asp-96 is deprotonated and the retinal chromophore is in the trans configuration. Also, a cooperative mechanism of the production of Ms cannot be neglected at high excitation intensity. But the back-reaction N-M, which had been proposed to explain Ms, was shown to be insignificant at moderately high pH. Bacteriorhodopsin, a membrane protein found in Halobacterium halobium, functions as a light-energy-converting proton pump. Its polypeptide chain is folded into a conformation consisting of seven transmembrane a-helices, and the retinal chromophore is linked by a protonated Schiff base to Lys-216 which is buried within the interior of the protein (Henderson et al., 1990). When the pigment containing alltrans-retinal (bR570)l absorbs light, it undergoes a cyclic photoreaction that drives proton translocation across the membrane. This trans photocycle is approximated by the following scheme: bR570-K590-LSSO-hi410-N560-0640-bR570 (Lozier et al., 1975; Kouyama et al., 1988; Fodor et al., 1988). The initial step involves a trans-to-cis isomerization of the chromophore at the C13-Cl4 double bond. In the L-M transition, the Schiff base deprotonates and Asp-85 mediates proton transfer from the Schiff base to the extracellular membrane surface. In the M-N transition, the chromophore is reprotonated from Asp-96, which in turn receives a proton from the cytoplasmic side. The chromophore reisomerizes to the all-trans configuration and the protein relaxes to the initial state. Supported by research grants for "Biodesign Research Program" from RIKEN and "Solar Energy Conversion by Means of Photosynthesis" from the Agency of Science and Technology of Japan and partly by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan.