Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Proteorhodopsin (PR), a light-driven proton pump from marine proteobacteria, exhibits photocycle characteristics similar to bacteriorhodopsin (BR) at neutral pH, including an M-like photointermediate. However, at acidic pH, spectroscopic evidence for an M-like species was absent, and the vectoriality of proton pumping was inverted. To gain further insight into this unusual property, we examined the voltage dependence of stationary and laser flash-induced photocurrents of PR under different pH conditions upon expression in Xenopus oocytes. The current–voltage curves were linear under all conditions tested, and photocurrent reversal potentials distinctly depended on the pH gradient. PR mutants D97N and D97T exhibited transient and stationary inward currents already at neutral pH, showing that neutralization of the proton acceptor abolishes forward pumping and permits only inward proton transport. Mutation E108G, which disrupts the donor site for Schiff base (SB) reprotonation, resulted in largely reduced photocurrents, which could be strongly stimulated by azide, similar to previous observations on BR mutant D96G. When PR and BR photocurrents in response to blue or green laser flashes during or after continuous illumination were compared, direct electrical evidence for the occurrence of an M-like intermediate at neutral pH could only be obtained when reprotonation of the SB was slowed down by PR mutation E108G. For PR at acidic pH, laser flashes only produced inwardly directed photocurrents, independent from background illumination, thus precluding electrical identification of an M-like species. However, when visible absorption spectroscopy was carried out at low temperatures, occurrence of an M-like species was robustly observed at low pH. This indicates that SB deprotonation and reprotonation occur during the PR photocycle also at low pH. Our results corroborate the conclusion that in PR, the direction of proton pumping can be switched by changes in pH and membrane potential, with the protonation state of Asp-97 being the key determinant for selecting between transport modes.