**3.8 Importance of the method for p***K***<sup>a</sup> estimation of crucial residues involved in proton transfer**

As described previously, sequential proton transfer events during the photocycles in various microbial H<sup>+</sup> -pumping rhodopsins, including BR, are successfully accomplished by regulating rigorous p*K*<sup>a</sup> changes among the crucial residues (particularly, PSB (or deprotonated SB), its proton acceptor, donor, and known or unknown proton-releasing residue(s)) related to proton translocation. The estimation of the p*K*<sup>a</sup> values of these residues in both unphotolyzed and photolyzed states, therefore, provides important clues for understanding the proton transfer mechanism in these H<sup>+</sup> pumps. Such p*K*<sup>a</sup> values can be indirectly estimated using spectroscopic approaches, such as FTIR or NMR. However, the establishment of a more direct method for p*K*<sup>a</sup> estimation is preferable, which can be achieved by measuring the photoinduced proton exchange between the protein and media (proton uptake/release) arising as a result of proton transfer events during the photocycle at varying pH values.

As a method for measuring proton movement transiently occurring during the photocycles of these pigments, the conventional method of using various pH-indicator dyes is frequently employed [44]. This method is highly time-resolved because the transient pH changes of the media with photoinduced proton uptake and release in rhodopsins are monitored based on the real-time transient absorbance changes of these pH-sensitive dyes in the sample suspension. The use of this method, therefore, enables us to precisely identify the timing of proton uptake and release together with the rise and decay kinetics of photoproducts. However, the pH range for measurement is confined to the pH values around its p*K*a; therefore, p*K*<sup>a</sup> estimation using this method is difficult. In contrast, another method using a tin oxide (SnO2 or indium-tin oxide, ITO) transparent electrode [160–163] is also highly pH-sensitive and rapidly time-resolved, although the applicable time period is limited within the time scale from several ten to hundred microseconds to hundreds of milliseconds [162, 164]. Moreover, this method can detect small pH changes with photoinduced proton uptake and release in the vicinity of a protein-attached electrode as a sufficiently large amplitude of voltage changes, even in solutions containing a small amount of buffer agents. Based on these advantages, we applied this method to the p*K*<sup>a</sup> estimation in H+ -pumps from three biological kingdoms, BR, GPR, and ARII [130, 162, 164]. The p*K*<sup>a</sup> estimation was performed in two ways. One method estimates the p*K*<sup>a</sup> value from the pH dependence of the magnitude of voltage changes, reflecting proton uptake and release. **Figure 3A** shows the photoinduced transient proton transfers in GPR at varying pH values, where the upward and downward shifts represent proton release and uptake, respectively. Because the peak time and magnitude of these data depend on both the on- and off-time constants, the fraction of the subpopulation inducing proton uptake or release at the respective pH values is estimated by fitting with the kinetically derived theoretical equation. **Figure 3B** shows the plots of the values estimated from the above fitting analysis as a function of pH. From further fitting analysis for these plots with an equation developed based on the Henderson– Hasselbalch theory, we succeeded in estimating the p*K*<sup>a</sup> values of some residues associated with photoinduced proton transfer events in GPR [164]. In contrast, the direct plots of amplitudes of light-induced proton transfer signals without strict regression described above, which are approximately proportional to the amount of proton transfer, also exhibited similar pH-dependent behavior, although these plots include some error. Therefore, such plotting may be useful as a method for simply estimating the approximate p*K*<sup>a</sup> values. Another method estimates the p*K*<sup>a</sup> values from the pH dependence of the kinetics of photoinduced proton uptake or release. **Figure 3C** shows the pH-dependent changes in the traces of the part representing the latter proton uptake following the initial release of a proton in BR in the pH range of 6.5 9.5. The fitting for these traces with a single exponential equation (solid curves in this figure) gave the rate constant values of proton uptake at the respective pH values. Similarly, the estimated values of the rate constant at each pH were plotted against pH (**Figure 3D**), and sequentially, the p*K*<sup>a</sup> values of Asp96BR in H+ -uptake (N-O transition) were estimated using the Henderson–Hasselbalch Equation [162]. All other BR values estimated by these methods were consistent with the corresponding values previously estimated using other experimental approaches [45] (also see **Figure 4B**). Therefore, this method for estimating the p*K*<sup>a</sup> values of some crucial residues for proton pumping, which is an index of proton pumping efficiency, may be a powerful and effective tool for screening efficient H<sup>+</sup> -pumps or their engineered mutants for optogenetics.

### **4. Future perspectives of H<sup>+</sup> -pumping rhodopsins as optogenetic tools**

As described at the beginning of this chapter, outward H+ -pumping microbial rhodopsins can evoke stronger light-induced neural suppression and quicker recovery from the inactivated state formed upon illumination than inward Cl-pump HRs. Hence, optical neural control using H<sup>+</sup> -pumping rhodopsins may also be an effective alternative for optogenetics, although neural inhibition with light-gated anion channel ACRs has recently attracted attention. The rational design based on the functional molecular basis of these rhodopsins described in this chapter may allow the creation of "neo-type" H+ -pumping microbial rhodopsins by introducing several mutations to further enhance the effect of neural silencing upon illumination, resulting in the acceleration of the development of more efficient tools for optogenetics along with developing color variants with various spectral properties. Further increases in protein expression and stability in targeted neural cells could also lead to the improvement of optogenetic tools. For this purpose, taking advantage of the abundance of these H<sup>+</sup> pumping rhodopsins, the exploration of new microbial H<sup>+</sup> -pumping rhodopsins with novel properties (e.g., high thermal stability [165, 166]) from nature may be useful for producing mutants.

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

### **Figure 4.**

*Schematic representation of the photocycle and accompanying proton transfer of three types of H<sup>+</sup> -pumping rhodopsins. (A) Schematic diagram of the photochemistry of BR. The stepwise proton transfer reactions are depicted by thin blue arrows and overlaid on the crystal structure of BR at the dark state (PDB 1c3w). The timing of H<sup>+</sup> -release differs depending on pH values. The expected configuration changes of chromophore retinal (RET) and PSB in each photocycle intermediate are also depicted. (B) Summary of pKa changes in the residues participating in the proton transfer reactions during the photocycle. A transient pKa increase and decrease of respective residues upon each transition are shown in upward and downward thin arrows. The reverse of pKa values between two adjacent residues leads to a unidirectional proton movement from a (protonated) residue with a lowered pKa value to another (unprotonated) residue with an elevated pKa value. Such proton migrations are expressed in thick blue arrows. The values in parentheses represent our previous estimated pKa values by the SnO2 electrode method [162]. (C) Schematic diagram of the photochemistry of a eubacterial H<sup>+</sup> -pump GPR. The proton transfer reactions are the case in the pH region below ca. 9.5. H-X represents an unidentified residue whose deprotonation at the initial state induces the formation of another parallel photocycle via a different M-like state (Ma) from normal M and early proton release [164]. (D) Schematic diagram of the photochemistry of a eukaryotic H+ -pump ARII. The timing of H+ -release is divided into three patterns depending on pH [130]: 1) an initial H+ -release from an unknown Y-residue (H-Y) at pH <sup>&</sup>gt; 10 (shown in orange arrow), 2) an initial H+ release from Glu199ARII at 7.5 <sup>&</sup>lt; pH <sup>&</sup>lt; 10 (shown in blue arrow), and 3) a probably direct H+ -release from Asp81ARII at the latter stage (O-ARII' transition) of the photocycle (shown in gray arrow). In panels C and D, the pKa values of several crucial residues for H<sup>+</sup> -pumping in respective rhodopsins, which were previously estimated using the SnO2 (or ITO) electrode method [130, 145, 164], are shown together.*

In addition, studies on H+ -pumping microbial rhodopsins are required to develop novel optical cellular control methods because these types of pigments can simultaneously induce alkalization of the intracellular pH by illumination-induced outward proton transport. As is generally known, the maintenance of an appropriate cellular pH is necessary to ensure that each requisite enzyme for various biological reactions functions properly. Because the drainage of acids produced by cellular metabolism is controlled through the Na<sup>+</sup> /H+ antiporter or the Cl/HCO3 exchanger to maintain the cellular pH near neutral, failure in these transporting systems affects the normal function of cells. Therefore, the application of optogenetics to cells with abnormal pH values, that is, light-induced manipulation of the cells specifically expressing H+ pumping microbial rhodopsins, may allow the restoration of the functions of these cells. As an example of intracellular pH regulation by optogenetics, Matsui et al. reported that the photoinduced intracellular pH increase in glial cells expressing aR-3 (Arch) suppressed the release of glutamate from these cells, which was triggered by glial acidosis upon brain ischemia, thereby ameliorating the effects of ischemic brain damage [167]. Moreover, the optical regulation of the function of varying organelles expressing H<sup>+</sup> -pumping rhodopsins has recently been attempted. Rost et al. demonstrated that selective Arch expression on synaptic vesicles together with a pHsensitive indicator and successive illumination led to vesicular acidification via Arch instead of vacuolar-type H<sup>+</sup> -ATPases (V-ATPases), enabling neurotransmitter accumulation within synaptic vesicles driven by the proton motive force (PMF) generated through light-activated Arch [168]. In addition, Hara et al. achieved dR-2-mediated optical partial suppression of cell death induced by the inhibition of respiratory PMF generation in the mitochondria of mammalian cells [169]. More recently, a method for topological inversion of microbial rhodopsins as optogenetic tools was also developed [170]. Hence, the application of this technique together with the use of recently discovered natural inward H+ -pumping rhodopsins [171, 172] as optogenetic tools may allow the induction of both light-activated acidification and alkalization in various types of cells or organelles such as mitochondria, vesicles, and lysosomes. Hence, the combination of an outward H+ -pumping rhodopsin and the topological reversal technique described above may allow various types of optogenetics. For instance, the use of outward H+ -pumping rhodopsin might lead to the following optogenetics: in general, the pH values of lysosomes in normal cells are regulated to be approximately 5, whereas those of lysosomes in cancer cells with acquired resistance to carcinostatic agents tend to be lower [173, 174]. The efficacy of carcinostatic agents for these cancer cells is degraded because they get trapped in acidified organelles; therefore, specific expression of H<sup>+</sup> -pumping rhodopsins in lysosomes of drug-resistant cancer cells and optical pH control (photoinduced alkalization) of these cellular organelles might lead to the restoration of the original effect of drugs. Thus, optogenetics using H+ -pumping microbial rhodopsins may lead to the establishment of new optical therapies in the future.
