Time-Resolved Measurements of Photovoltage Generation by Bacteriorhodopsin and Halorhodopsin Adsorbed on a Thin Polymer Film

Abstract

We constructed a time-resolved photovoltage measurement system and examined the photovoltage kinetics of wild-type bacteriorhodopsin, its D96N mutant, and halorhodopsins from Halobacterium salinarum and Natronobacterium pharaonis. Upon illumination with a laser flash, wild-type bacteriorhodopsin showed photovoltage generation with fast (10-100 micros range) and slow (ms range) components while D96N lacked the latter, as reported previously [Holz, M., Drachev, L.A., Mogi, T., Otto, H., Kaulen, A.D., Heyn, M.P., Skulachev, V.P., and Khorana, H.G. (1989) Proc. Natl. Acad. Sci. USA 86, 2167-2171]. In contrast, photovoltage generation in halorhodopsins from H. salinarum and N. pharaonis was significant only in the ms time range. On the basis of the photovoltage kinetics and photocycle, we conclude that major charge (chloride) movements within halorhodopsin occur during the formation and decay of the N intermediate in the ms range. These observations are discussed in terms of the "Energization-Relaxation Channel Model" [Muneyuki, E., Ikematsu, M., and Yoshida, M. (1996) J. Phys. Chem. 100, 19687-19691].

Probing the Cl−-pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle

“…Thus, the membrane potential appears to have significant effects on the Cl − transfer in the CP channel compared to those in the EC channel. This result might be consistent with previous reports: Ludmann et al [15] and Muneyuki et al [19] detected much smaller electrogenicity during the NpHR′ formation compared to that during the O formation. These observations may reflect the difference in dielectric constants of CP and EC channels.…”

“…However, the previous flashinduced voltage measurements led to the conclusion that O does not appear in the Cl − transport photocycle [18]. On the other hand, other voltage measurements detected large electrogenicity during O formation [15,19]. These data support the model that Cl − release is associated with O formation.…”

“…A interior negative membrane potential hinders the inward movement of Cl − . Previous studies suggested that during the formation of N, Cl − moves from Site I to the CP channel over the Schiff base [15,19,20]. However, the formation of N, which probably corresponds to the P 2 to P 3 transition, did not slow down when a interior negative membrane potential was imposed (see k 2 values in Table 1).…”

Chloride Concentration Dependency of the Electrogenic Activity of Halorhodopsin

“…Recently, time-resolved photovoltage analyses were reported for phR (15,26) and shR (26). In both phR and shR, major charge movement was observed in the millisecond time scale, and it was proposed that N (and/or O) intermediate formation was associated with charge movement (26).…”

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis

“…HR is a light-driven, inward chloride pump associated with proton influx upon light activation (3,4), and the photocycle of HR includes the K, L, N, and O states (5,6). Among these series of intermediates, the process of chloride release occurs during the N-to-O-state transition (6)(7)(8)(9). It has been suggested that HR plays a significant biological role in either promoting the proton-motive force (10) to facilitate BR-assisted energy production, or maintaining a cellular osmolarity equilibrium (3,11) under the high-salinity environments found in their habitat.…”