Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH

Abstract

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.

Photochemical characterization of actinorhodopsin and its functional existence in the natural host

“…Finally, the H + release occurs again, and the signal returns to the baseline. Unexpected first H + release is also observed in PR, which also lacks PRG, at alkaline pH [35,37]. However, the H + release in RpActR appears not to reflect the same reaction in PR.…”

“…The flash-induced H + transfer was measured using an indium tin oxide (ITO) transparent electrode, which has been shown to act as a time-resolved pH sensor [35][36][37]. In this experiment, the PC-reconstituted RpActR was used.…”

“…Further insight into the photocycle was obtained from measurements using a transparent ITO electrode, which functions as a good time-resolved pH electrode [36,37].…”

“…However, the H + release in RpActR appears not to reflect the same reaction in PR. For PR, the first H + release is predicted to occur toward the CP medium, implying no contribution to the outward H + pumping activity [37]. For RpActR, the outward H + -pumping activity reaches its maximum value at pH 7, a physiological pH (data not shown).…”

“…This finding was confirmed by the light-induced pH change in the suspension of E. coli cells. Moreover, for PR, the first H + release is associated with the formation of another M intermediate, which forms in a branched photocycle appearing at alkaline pH [37]. This branch is observable above pH 8.5.…”