Light-Induced Proton Uptake and Release of <i>pharaonis</i> Phoborhodopsin Detected by a Photoelectrochemical Cell

Abstract

Phoborhodopsin (pR, also called sensory rhodopsin-II, sR-II) is a receptor for the negative phototaxis of Halobacterium salinarum (pR), and pharaonis phoborhodopsin (ppR) is the corresponding receptor of Natronobacterium pharaonis. 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 (∼50 μM)|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.