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	Figure 1. Basic characterization of CsR in Xenopus laevis oocytes and HEK-293 cells.(a) Proposed structure of CsR-WT with mutated residues based on the BR crystal structure (PDB: 1M0L). Brackets indicate respective positions in BR and demonstrate the high conservation of key residues. Arrows highlight the proposed proton transport pathway of CsR-WT, which is similar to BR. A dotted arrow is used to indicate that whether R83 deprotonates itself during the photocycle remains to be determined. (b) Typical photocurrents of CsR-WT measured in oocytes at extracellular pHo 7.5 after illumination with 550 ± 25 nm. Holding potentials were increased from −125 mV in 25 mV steps up to +75 mV. Separated current trace illustrates the biexponential decay after light illumination. (c) Action spectra measured in oocytes at pH 7.2 with supplementation of 5 μM all-trans retinal (ATR, n = 7) and without ATR (n = 5). Currents were normalized to the maximal stationary currents. Inset shows absolute amplitudes at 0 mV (ATR, n = 16; without ATR, n = 21). (d) Ion selectivity measured in oocytes at pHo 7.2 and 0 mV (all n = 5 except CsCl, n = 3). (e) Absolute photocurrent amplitudes of diverse microbial pumps at pHo 7.5 as measured in oocytes (all at 0 mV, illumination with 550 ± 25 nm, CsR [n = 24], Arch3 [n = 8], BR [n = 19], βHK-BR [n = 17], NpHR [n = 6]). (f) Absolute photocurrent amplitudes of CsR-WT-EGFP (n = 8) and Arch3-WT-EGFP (n = 11) measured in HEK-293 cells (0 mV, 550 ± 7 nm, pHo 7.2, pHi 7.2 after 24–30 h, 1 μM ATR). (g) Confocal images of HEK-293 cells transfected with CsR-WT-EGFP or Arch3-WT-EGFP (after 24–30 h, 1 μM ATR). Scale bars represent 25 μm. Data represent the mean ± SE.
	Figure 2. Current traces and IStat(E) of CsR-WT, CsR-D86T, and CsR-D211N at different extracellular pH values.(a) Photocurrents of CsR-WT were less dependent on pH and always outward directed (550 ± 25 nm). (b) IStat(E) of stationary photocurrents of CsR-WT (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 10], pH 7.5 [n = 25], pH 5 [n = 7]). (c) Photocurrents of CsR-D86T upon illumination with green light (550 ± 25 nm). Inward-directed peak currents were observed, especially at low pHo. These are followed by outward directed peaks immediately after illumination. (d) Action spectra show two separated protein populations of CsR-D86T. Both are shifted towards CsR-WT as shown by dotted lines. Spectrum of stationary currents was measured at pH 10 and 0 mV (n = 4). Spectrum of peak currents in green was measured at pH 7.5 and 0 mV (n = 4). (e) Stationary photocurrent traces of CsR-D86T upon illumination with blue light (400 ± 25 nm). Stationary currents disappear at low pH. (f) IStat(E) of stationary currents of CsR-D86T (400 ± 25 nm, normalized to pH 10 and 0 mV, pH 10 [n = 13], pH 7.5 [n = 8], pH 5 [n = 5]). (g) Photocurrents of CsR-D211N are directed outward and inward at high and low pHo, respectively (550 ± 25 nm). (h) IStat(E) of stationary photocurrents of CsR-D211N (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 6], pH 7.5 [n = 7], pH 5 [n = 6]). Data were measured in oocytes and represent the mean ± SE.
	Figure 3. Current traces and IStat(E) of CsR-R83Q and CsR-R83Q-D86N at different extracellular pH values.(a) Photocurrents of CsR-R83Q are characterized by strong inward-directed photocurrents at low pHo (550 ± 25 nm). (b) IStat(E) of stationary photocurrents of CsR-R83Q (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 7], pH 7.5 [n = 10], pH 5 [n = 6]). (c) Photocurrents of CsR-R83Q-D86N (550 ± 25 nm). (d) IStat(E) of CsR-R83Q-D86N stationary photocurrents (550 ± 25 nm, normalized to pH 5 and 0 mV, pH 7.5 [n = 7], pH 5 [n = 9], pH 4 [n = 5]). Double mutant shows that inward-directed channel-like currents are independent of the primary proton donor D86. (e) Stationary photocurrent traces of CsR-R83Q-D86N upon illumination of blue light (400 ± 25 nm). Similar to D86N, low outward-directed stationary currents were observed at high pHo. (f) IStat(E) of CsR-R83Q-D86N stationary photocurrents (400 ± 25 nm, normalized to pH 10 and 0 mV, pH 10 [n = 5], pH 7.5 [n = 4], pH 5 [n = 5]). Data were measured in oocytes and represent the mean ± SE.
	Figure 4. Current traces and IStat(E) of CsR- Y57K and CsR-Y57K-R83Q at different extracellular pH values.(a) Photocurrents of CsR-Y57K are characterized by strong outward-directed photocurrents at high pHo (550 ± 25 nm). (b) IStat(E) of stationary photocurrents of CsR-Y57K (550 ± 25 nm, normalized to pH 7.5 and +75 mV, pH 10 [n = 6], pH 7.5 [n = 9], pH 5 [n = 4]). (c) Photocurrents of CsR-Y57K-R83Q exhibit the properties of both single mutations but without huge amplitudes at high electrochemical loads (550 ± 25 nm). (d) IStat(E) of stationary photocurrents of CsR-Y57K (550 ± 25 nm, normalized to pH 5 and 0 mV, pH 10 [n = 9)], pH 7.5 [n = 9], pH 5 [n = 9]). Data were measured in oocytes and represent the mean ± SE.
	Figure 5. Inward-directed photocurrents produced by CsR-R83Q are channel-like.(a) Position W182 is highly conserved in microbial rhodopsins and is located close to the methyl groups of the retinal. (b) Photocurrents of CsR-W182F are characterized by a large transient outward peak compared to the stationary currents. (c) IStat(E) of stationary photocurrents of CsR-W182F are always directed outward but curved compared to CsR-WT (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 4], pH 7.5 [n = 6], pH 5 [n = 5]). (d) Similar to CsR-R83Q, photocurrents of CsR-R83Q-W182F are inward-directed, especially at low pHo. Additionally, the currents show drastically increased decay times. Data were measured in oocytes and represent the mean ± SE.
	Figure 6. Molecular dynamics simulation and prediction of water molecule distribution of dark states and O-intermediates of BR.(a) The dark state of BR-WT with separated areas of mobile water. (b) In the O-state of BR-WT, the downward (extracellular side) movement of the guanidine side chain of R82 is depicted. The hydrogen bond connecting the guanidine group with Y57 and D212 was interrupted due to this downward movement. (c) Mutation of R82 by the smaller and neutral Q82 induced a large space with an extended water cluster. (d) Extended water cluster was still present in the O-state of R82Q, but direct accessibility to the primary proton donor D85 was interrupted. (e) Mutation of Y57 by the positively charged K57 promoted a salt bridge between D212 and K57. The dark state of K57Y was also characterized by an increased water cluster. (f) The O-state of Y57K lacks an interaction between R82 and E204. The huge cavity of mobile water molecules between K57, R82, D85, and D212 caused accessibility to the Schiff Base. Downward movement of the guanidine side chain of R82 was also observed.
	Figure 7. Reversal voltages of Δp- and ΔΨ-type light-driven proton pumps.Reversal voltage (Erev) of Gloeobacter rhodopsin (GR), CsR-WT, CsR-T46N, CsR-R83Q, and CsR-R83Q-T46N are plotted against the extracellular pHo. The black line represents the theoretical expected Erev of free proton flow calculated by the Nernst equation based on an intracellular pHi of 7.4. Reversal potentials below this line indicate proton pump activity. Values from −125 mV up to +75 mV were determined directly. Other values are determined by extrapolation. CsR-WT (550 ± 25 nm, pH 10 [n = 8], pH 9 [n = 3], pH 7.5 [n = 26], pH 6 [n = 5], pH 5 [n = 8], pH 4 [n = 6]) exhibited high resistance against low pHo whereas GR (400–600 nm, pH 7.5 [n = 23], pH 6 [n = 6], pH 5 [n = 9], pH 4 [n = 10]) was shifted −260 mV in parallel to the calculated potentials. Mutation of CsR-R83 (550 ± 25 nm, pH 10 [n = 7], pH 9 [n = 4], pH 7.5 [n = 11], pH 6 [n = 4], pH 5 [n = 6], pH 4 [n = 4]) into Glu converts Coccomyxa rhodopsin into a Gloeobacter-like proton pump in relation to the Erev. CsR-T46N (550 ± 25 nm, pH 10 [n = 4], pH 7.5 [n = 8], pH 5 [n = 4], pH 4 [n = 4]). CsR-T46N-R83Q (550 ± 25 nm, pH 10 [n = 4], pH 7.5 [n = 5], pH 5 [n = 5], pH 4 [n = 2]). Data were measured in oocytes and represent the mean ± SE.

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