Light-induced proton uptake and release of pharaonis phoborhodopsin detected by a photoelectrochemical cell

Phoborhodopsin (pR, also called sensory rhodopsin-IT, sR-II) is a receptor for the negative phototaxis of Halobacterium salinarum (pR), and pharaonis phoborhodopsin (ppR) is the corresponding receptor of Natronobacterium pharonis. pR and ppR are retinoid proteins and have a photocycle similar to that of bacteriorhodopsin. The photocycle of ppR in the millisecond time range includes M and O intermediates: ppR --> M --> O --> ppR. A photoelectrochemical cell was constructed composed of SnO2\thin ppR solution (similar to 50 mu M)\400 mM NaCl\SnO2. Photoinduced potential differences between two SnO2 transparent electrodes were measured. They were caused by changes in pH close to the SnO2 electrode surface. The signal was time-differentiated to envisage the direction of pH change and proton movement. A positive signal was due to a decrease in the local pH, i.e., proton release from ppR, and a negative signal was caused by the proton uptake. Immediately upon irradiation with continuous light, the transient negative on-response was observed for all pH examined. The shape of the off-response on turning off the light was pH-dependent: at alkaline or neutral pH, a negative component was observed followed by a positive component. The off-response was measured after the photosteady state was attained. The shape of the off-response well correlated with the ratio of contents of the M and O intermediates at the steady state. It is concluded that the proton uptake occurs during M --> O and the proton release during O --> ppR transitions.