**5. Conclusion**

Proton pump-type microbial rhodopsins are not only effective neural suppressors but also optical tools for pH control of various cells or organelles that specifically incorporate these pigments, which makes them a dual optogenetic tool. Rational protein engineering based on molecular mechanisms is required to further develop these rhodopsins into more effective tools. Considering the photochemical reaction

*Functional Mechanism of Proton Pump-Type Rhodopsins Found in Various Microorganisms… DOI: http://dx.doi.org/10.5772/intechopen.97589*

and accompanying proton transfer mechanism in various H<sup>+</sup> -pumping rhodopsins described previously, mutations that increase their photocycle kinetics may be effective for enhancing the respective H+ -pumping abilities. To increase their H+ -pumping efficiency via their photocycles, for example, a mutation that lowers the p*K*<sup>a</sup> values of proton acceptors in the unphotolyzed state, which increases the population with H<sup>+</sup> pumping activity, may be effective. Alternatively, alterations that lead to a reduction in the p*K*<sup>a</sup> of the proton donor upon M–N transition (donor-to-SB H+ -transfer) and to an increase in its p*K*<sup>a</sup> value upon N–O transition (H+ -uptake) may be efficacious for promoting CP-side proton transfer. In addition, the introduction of PRC-forming residues on the EC surface may facilitate EC proton transfer. While screening for more effective tools among such designed mutants based on their molecular mechanism, the SnO2 (ITO) electrode method could be a simple and efficient tool for estimating the p*K*<sup>a</sup> values of critical residues for proton pumps, which is an index of proton pumping effectiveness. Thus, through a series of investigations on H<sup>+</sup> pumping rhodopsins based on molecular mechanisms, novel optogenetic H+ -pumping rhodopsins could be developed in the near future.
