A Study on Mechanisms Underlying Proton Transport in Proton Pump-type Microbial Rhodopsins

Abstract

Microbial rhodopsins are photoreceptive membrane proteins composed of seven transmembrane α-helical apoproteins (opsin) and a covalently bound retinal chromophore. Microbial rhodopsins exhibit a cyclic photochemical reaction referred to as photocycle when illuminated. During their photocycles, these proteins perform various functions such as ions transport and photosensing. Among the various functional types of rhodopsins found to date, we have focused on the utility of proton pump-type microbial rhodopsins as optogenetic tools for optical pH control in cells or organelles. To develop effective toolkits for this purpose, a deeper understanding of the proton-pumping mechanism in these rhodopsins may be required. In this review, we first introduce a useful experimental method for measuring rapid transient pH changes with photoinduced proton uptake/release using transparent tin oxide (SnO2) or indium-tin oxide (ITO) electrodes. In addition, we describe the unique pH-dependent behavior of the photoinduced proton transfer sequence as well as the vectoriality of proton transportation in proteorhodopsin (PR) from marine eubacteria. Through intensive ITO experiments over wide pH range, including extremely high or low pH values, in combination with photoelectric measurements using Xenopus oocytes or a thin polymer film "Lumirror," we encountered several interesting observations on photoinduced proton transfer in PR:1) proton uptake/release sequence reversal and potential proton translocation direction reversal under alkali conditions, and 2) fast proton release from D227, a secondary counterion of the protonated retinal Schiff base at acidic pH values.