**3.4 Initial proton transfer from PSB to the proton acceptor, aspartate, upon L–M transition: the most crucial step for proton transport**

As described above, the proton acceptor residue from PSB corresponding to Asp85BR is superconserved in all H+ -pump-type rhodopsins, suggesting the significance of this residue in the proton pumping mechanism. The negative charge of deprotonated Asp85BR interacts with another deprotonated aspartate Asp212BR and three water molecules through hydrogen bonds, forming a pentagonal cluster that electrostatically stabilizes two positive charges of PSB and Arg82BR [47]. The same cluster structure has also been observed in H+ -pumping rhodopsins other than BR [22, 131]. In this sense, two aspartates also play an important role in counterions to PSB, in which Asp85BR and Asp212BR are referred to as primary and secondary counterions, respectively. The aspartate residue, which is the proton acceptor, is deprotonated in the unphotolyzed state under physiological conditions. At pH values below the p*K*<sup>a</sup> of the proton acceptor in the resting state, where this residue takes the protonated form, initial proton migration from PSB does not occur; thus, the formation of the M state with deprotonated SB is not observed and H<sup>+</sup> -pumping activity vanishes. The p*K*<sup>a</sup> of the proton acceptor in the unphotolyzed state, therefore, tends to adopt as low a value as possible, for example, ca. 2.5 for Asp85BR [134]. Asp85BR is conjugated with PRC located on the EC surface, which contributes to the retention of its low p*K*<sup>a</sup> in the dark state [45, 134].

In contrast, the p*K*<sup>a</sup> values of proton acceptor residues in eubacterial H+ -pumps tend to be relatively higher, for example, approx. 7-7.5 for GPR [105, 106, 109, 113], 7.8 (or 6.2) for BPR [113], 6.0 for XR [135], 4.5 for GR [136], 6.0 for ESR [137], and 5.8 for ActR [116]. Such high p*K*<sup>a</sup> values in these pigments are thought to be associated with

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

physiological pH conditions of the hosting bacteria possessing these rhodopsins; because the habitat of bacteria containing PR-like proteins (e.g., sea water, freshwater, etc.) usually has near-neutral or weakly alkaline pH conditions (approx. pH 6.5-8.5) that are above the p*K*<sup>a</sup> values of the proton acceptors in the dark state, the proton acceptors of these H+ -pumps can adopt the deprotonated form to work as proton pumps. The elevated p*K*<sup>a</sup> values of the proton acceptors in eubacterial-type H+ -pumps may be due to the absence of two EC glutamates corresponding to Glu194BR and Glu204BR and the repositioning of an arginine residue (corresponding to Arg82BR) located within the pentagonal cluster in the EC channel [138]. Moreover, in GPR, it was clarified that a highly conserved histidine residue His75GPR among bacterial H+ -pumps that is adjacent to the proton acceptor Asp97GPR contributes to the adjustment of the higher p*K*<sup>a</sup> of Asp97GPR in the unphotolyzed state because the mutation of this residue significantly decreases the p*K*<sup>a</sup> of Asp97GPR [139]. The replacement of the corresponding histidine residues in other eubacterial H+ -pumps, however, did not cause such a large change in the p*K*<sup>a</sup> of their proton acceptor residues [136, 137], implying that the above-mentioned p*K*<sup>a</sup> modulation mechanism through histidine is not common in all eubacterial-type H+ -pumps.

The primary and secondary counterions (corresponding to Asp85BR and Asp212BR, respectively) are located near and arranged symmetrically around the PSB, resulting in forming a part of the proton acceptor cluster. The secondary counterion is also deprotonated like the primary counterion (proton acceptor) because the p*K*<sup>a</sup> of this residue in the resting state usually takes a further lower value compared to the primary counterion. Nevertheless, a proton of PSB is always transferred to the primary counterion at the photoproduct rather than the secondary counterion. How should this proton transfer mechanism be considered? In the case of BR, it is thought that upon L–M transition, the p*K*<sup>a</sup> of PSB is lowered from a value above 13 in the dark state to a value below 3, which is near the p*K*<sup>a</sup> (2.5 [134]) of Asp85BR in the same state. In contrast, the p*K*<sup>a</sup> of Asp85BR simultaneously increases to a value of at least 8.5 approximately at this time (the first increase in the p*K*<sup>a</sup> of Asp85BR) [140]. Thus, the p*K*<sup>a</sup> values between PSB and Asp85BR are reversed, giving rise to a one-way proton movement from PSB to the deprotonated Asp85BR. Then, the p*K*<sup>a</sup> of Asp85BR finally increases to above 10 in the M-state, thus allowing it to maintain its protonated state until the end of the photocycle (a second increase in the p*K*<sup>a</sup> of Asp85BR) [140]. The second increase in the p*K*<sup>a</sup> of Asp85BR upon M-formation is thought to be triggered by the disruption of the electrostatic interaction between the negatively charged Asp85BR and the positively charged Arg82BR in PRC, which is caused by the protonation of Asp85BR and the accompanying deprotonation of PRC (initial proton release from PRC) during this process [140].

In contrast, a question that could arise would be how p*K*<sup>a</sup> regulation in the proton acceptors of PR-like eubacterial H<sup>+</sup> -pumps lacking their coupled PRC is achieved. Although there is no experimental evidence, we may presume that a similar p*K*<sup>a</sup> inversion between PSB and its proton acceptor (Asp97GPR) in BR occurs upon the formation of M in GPR; the p*K*<sup>a</sup> of PSB decreases from > 11 in the unphotolyzed state [141] to 3 upon M-rise, resulting in it being lower than the p*K*<sup>a</sup> of Asp97GPR in the dark state (7-7.5). The possibility of an increase in the p*K*<sup>a</sup> of Asp97GPR in the M-state similar to BR has also been reported [142]. FTIR data in DMPC-reconstituted vesicles revealed that the origin of the first proton release upon M-rise observed in GPR under alkaline conditions (pH 9.5) is not Asp97GPR, which is protonated during this transition [142]. This observation implies that the p*K*<sup>a</sup> of Asp97GPR at M is above 9.5. Why is the photoinduced p*K*<sup>a</sup> increase in Asp97GPR caused by the absence of a

BR-like interaction with PRC? Although the reason is still unclear, an alternative interaction with neighboring His75GPR [143] may work instead of the PRC, which is missing in GPR.

Through low-temperature FTIR experiments, it was suggested that PSB forms a stronger hydrogen bond with Asp227GPR rather than Asp97GPR within the pentagonal cluster around PSB upon K-formation [144]. In addition to this observation, the p*K*<sup>a</sup> of Asp227GPR in the unphotolyzed state was estimated to be approximately 2.6 or 3.0 [141, 145]. Hence, we cannot exclude the possibility that Asp227GPR receives a proton from PSB at the photoproduct under such low pH conditions (3 < pH < 7), where Asp97GPR and Asp227GPR are protonated and deprotonated at the resting state, respectively. Our experimental data using a rapid time-resolved pH-sensitive electrode method (described later with the details of this experimental method), however, showed that the p*K*<sup>a</sup> of Asp227GPR may further decrease from 3 at the dark state to 2.3 at the photolyzed state [145]. This possible p*K*<sup>a</sup> decrease in Asp227GPR at the photoproduct might hinder its proton acceptance from the PSB. Even though Asp227GPR can transiently receive a proton from PSB, the proton might be immediately released to other dissociable residue(s) or internal waters. Interestingly, the computational calculations performed by Bondar et al. suggested that among three possible pathways of proton transfer from PSB to Asp85BR, that is, 1) a direct pathway to Asp85BR on the Thr89BR side of the retinal, 2) a proton wire through Thr89BR, and 3) a proton transfer pathway via Asp212BR, the energy barrier of the third proton transfer pathway was the smallest [146]. Thus, a similar photoinduced p*K*<sup>a</sup> decrease in the second counterion to GPR occurs even in other H<sup>+</sup> -pumping rhodopsins, including BR, and might play a role in the initial proton movement from PSB to its proton acceptor upon light activation of these pigments. Further studies are required to clarify the roles of this mechanism.

### **3.5 Diverse proton transfer occurring on the CP side**

Following the EC proton transfer in the first half of the photocycle, the CP proton transfer events via the SB proton donor in the second half of the photocycle after Mdecay are the next critical steps. The proton transfer mechanism at this stage varies among the three types of H+ -pumping rhodopsins—archaeal, bacterial, and eukaryotic. In the latter half of the BR photocycle, the deprotonated SB first accepts a proton from its proton donor, Asp96BR, located in the CP channel during the M–N transition. The p*K*<sup>a</sup> of SB in this reprotonation process was estimated to be approximately 8 [147]. In contrast, the p*K*<sup>a</sup> of Asp96BR is maintained at a higher value (<sup>&</sup>gt; 11) through an interhelical hydrogen bond with Thr46BR on the B-helix [148]. Therefore, the p*K*<sup>a</sup> of Asp96BR needs to be lower than the p*K*<sup>a</sup> value (8) of SB to release a proton toward deprotonated SB, from > 11 at the initial state to 7-7.5 [149, 150]. This p*K*<sup>a</sup> decrease is caused by the entry of water with the opening of the intracellular segment via the outward tilt of the F-helix at the M-state, leading to the internal hydration of the CP region. The inflow water breaks the interaction between Asp96BR and Thr46BR, facilitating hydrogen bonding rearrangement so that Asp96BR forms a new interaction with neighboring water chains [151]. Then, during the following N–O transition, the <sup>p</sup>*K*<sup>a</sup> value of Asp96BR increases again and finally reaches a higher value (<sup>&</sup>gt; 11) close to one in the dark state. Therefore, Asp96BR can capture a proton from the CP medium to reprotonate.

As described above, in GPR, the residue corresponding to Asp96BR is the conservative carboxylate, Glu108GPR. This residue can function as a proton donor to SB;

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

however, the proton movement from Glu108GPR to SB and the subsequent reprotonation of Glu108GPR from the CP bulk are indistinguishable, unlike BR; two sequential proton transfer events in the CP channel concurrently take place upon the M–N transition [105]. The difference in CP proton migration in eubacterial H+ pumps, including PR from BR, seems to be related to the difference in the environment around the proton donor in the intracellular part of the protein between them. In many eubacterial H+ -pumping rhodopsins, the interhelical hydrogen bonding pair corresponding to the Asp-Thr interaction in BR is replaced by the Glu-Ser interaction. The X-ray crystal structure of XR in the dark state revealed that the proton donor (Glu107XR) in the CP channel connects to the peptide carbonyl of the lysine residue (Lys240XR) in SB; therefore, the CP H-bonded chain via water is already formed in the unphotolyzed state [118]. Thus, the difference in the CP proton transfer scheme from BR may be due to the formation of the hydrophilic CP pathway in eubacterial H+ -pumps.

We also observed a further interesting characteristic in the CP proton transfer of the PR-like H<sup>+</sup> -pump ESR. The residue positioned at the site of the proton donor in ESR is the cationic residue Lys96ESR (see **Figure 2**). Nevertheless, Lys96ESR seems to be involved in the CP proton transfer from the intracellular aqueous space to the inner deprotonated SB because the replacement of this residue by other nonionizable residues resulted in a significant delay of the M-intermediate [114]. This observation exploded a conventional concept, the so-called carboxyl rule, that the functional proton-donating residue is confined to two carboxylates (Asp or Glu). Some distinct structural features of BR can be observed in the X-ray crystal structure of the ESR. One of the differences is the presence of a cavity around Lys96ESR located close to the CP bulk media [122]. Although Lys96ESR is surrounded by hydrophobic residues in the CP channel in the dark state similar to BR, the cavity in the vicinity of Lys96ESR is separated only by a polar side chain of Thr43ESR (corresponding to Phe42BR), in contrast to BR, whose proton donor residue is completely separated from the CP bulk solvent by a hydrophobic barrier composed of multiple hydrophobic residues including Phe42BR [122]. Connectivity with the CP bulk facilitates direct access of the protons from the CP solvent in Lys96ESR. Another difference is the flexibility of the side chain of Lys96ESR, which may allow the smooth repositioning of this residue by donating to SB and reprotonation. Given that these structural properties are present in the CP region together with the time-resolved spectroscopic data using D2O, it may be plausible that the CP proton transfer scheme in ESR is as follows [114]: Lys96ESR adopts an unprotonated form at the resting state to be buried within the hydrophobic CP region. Upon M-decay, Lys96ESR transiently catches a proton from the CP bulk solvent (at M1\$M2), and then, a little later, it donates a proton to SB (at M2\$N1). Hence, Lys96ESR acts as a residue facilitating proton delivery from the CP bulk to the SB, which is an apparently different proton donating mechanism from the conventional one.

Another unique example of CP proton transfer was found in two types of gramnegative rod-shaped Proteobacteria in soil: *Pseudomonas putida* rhodopsin (PspR) from *Pseudomonas putida* and *Pantoea ananatis* rhodopsin (PaR) from *Pantoea ananatis*, a plant pathogen [152]. The notable properties of these types of rhodopsins are the replacement of the residue corresponding to Asp96BR with nonionizable glycine and the presence of a specific histidine at the position corresponding to Thr46BR. This histidine residue is highly conserved in a member of this group and is assumed to constitute a part of a proton-donating complex [152]. However, it was observed that the rate of M-decay linearly depends on the proton concentration of the medium in a

### **Figure 3.**

*pKa estimation of critical residues for a proton pump by the SnO2 (or ITO) electrode method. (A) Photoinduced voltage changes representing proton uptake/release at varying pH values [164]. Noisy and smooth curves represent the observed and fitted curves, respectively. For fitting, we employed the following kinetic equation:* <sup>Δ</sup>*Voltage* <sup>∝</sup><sup>Δ</sup> *<sup>H</sup>*<sup>þ</sup> ½ �¼�*<sup>A</sup> kf*,*<sup>u</sup> ks*,*r*�*kf*,*<sup>u</sup> <sup>e</sup>*�*kf*,*ut* � *<sup>e</sup>*�*ks*,*rt* � � <sup>þ</sup> *<sup>B</sup> kf*,*<sup>r</sup> ks*,*u*�*kf*,*<sup>r</sup> <sup>e</sup>*�*kf*,*rt* � *<sup>e</sup>*�*ks*,*ut* � �*, where A represents a constant reflecting the fraction of the subpopulation photoinducing the first proton uptake followed by release and the rate constants of the first H+ -uptake and second H+ -release in such a proton transfer sequence are expressed by kf,u and ks,r, whereas B represents a constant reflecting the fraction of subpopulation inducing the opposite sequence of proton transfer, and the rate constants of the first H+ -release and second H+ -uptake in that case are expressed by kf,r, and ks,u, respectively. At pH < 9.5, where the first H+ -release cannot be obviously observed, it was assumed that B is almost zero. In contrast, the fitting at pH > 9.5 was conducted as A = 0. Six buffer agents with different pKa values were added to the media for experiments so that the buffering action remained constant over a wide pH range (*�*<sup>5</sup>* <sup>≤</sup> *pH* <sup>≤</sup> �*11). (B) Plot of amplitude of H+ -transfer versus pH. Filled and empty circles indicate plots of strict values with theoretical regression (*�*A and B values obtained by the above fitting) and approximate peak values of photoinduced signals estimated by sight, respectively. These values were plotted as relative values. A solid curve represents a curve fitted using the following equation:* <sup>Δ</sup> *<sup>H</sup>*<sup>þ</sup> ½ �¼�*<sup>C</sup>* <sup>1</sup> <sup>1</sup>þ10*pKa*1�*pH* � � <sup>1</sup> <sup>1</sup>þ10*pH*�*pKa*<sup>2</sup> � � � <sup>1</sup> <sup>1</sup>þ10*pKa*2�*pH* h i � � *, where C, pKa1, and pKa2 represent a scaling constant for the amplitude, pKa values of Asp97GPR, and an unidentified X-residue at the unphotolyzed state, respectively. The idea for the derivation of the equation has been described previously [164]. (C) Plots of the part of the second H+ -uptake after initial H+ -release as a function of time [162]. All values obtained at varying pH values (*◇*, pH 7.1;* ▽*, pH 7.5;* △*, pH 7.9;* □*, pH 8.4;* ○*, pH 9.0) were plotted as relative values. Continuous curves are fitting curves with single exponential eqs. (D) pH dependence of the rate constants of the second H+ -uptake (ku). Increments of ku at each pH obtained by subtraction of the minimum value at the highest pH were plotted as relative values. Filled and empty circles represent the plots for BR and ARII, respectively. These plots were well fitted with the Henderson–hasselbalch equation with a single pKa value. Respective fitting curves for BR and ARII are shown using solid and broken curves. Panels A and B were adapted with permission from Tamogami et al. [164], Biochemistry copyright 2016 American Chemical Society, whereas panels C and D were adapted with permission from Tamogami et al. [162], Photochem. Photobiol. Copyright 2009 the authors, journal compilation, the American Society of Photobiology.*

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

homologous protein in the same group [153], implying that the histidine forms a CP conductive channel rather than a proton-donating complex to enable rapid proton movement from the CP surface. Identification of the role of this unique histidine requires further study.

Among eukaryotic H<sup>+</sup> -pumps, both fungal and algal H+ -pumps possess the same proton donor aspartate residue as BR. For two algal H<sup>+</sup> -pump homologs ARI and ARII, however, the residue corresponding to Thr46BR is replaced by asparagine, which may cause different interactions with the proton donor and its p*K*<sup>a</sup> regulation from BR and fungal-type rhodopsins. Our experimental data indeed revealed that the p*K*<sup>a</sup> values of their proton donors (Asp100ARI and Asp92ARII) in H+ -uptake (N-O transition) are 6 (**Figure 3D**), which is ca. 1-1.5 units lower than that of BR (7-7.5) [130]. In the sensorlike fungal rhodopsin NR, the residue corresponding to Asp96BR is glutamic acid, similar to numerous eubacterial H+ -pumping rhodopsins, while the corresponding residue in the H+ -pump LR is aspartic acid, similar to BR. Interestingly, the substitution of the proton donor Asp150LR with an NR-like glutamate abolished the fast H<sup>+</sup> pumping photocycle [126, 127], implying that residues other than native aspartate work improperly in fungal H+ -pumps, even though it is a conservative one. In contrast, the influence of Asp-Glu replacement in Asp96BR differed depending on the experimental conditions [97, 154]. In the reconstituted BR heterogeneously expressed in *Escherichia coli*, the replaced glutamate residue fully functioned as a proton donor [97], whereas the replacement of BR in the native membrane led to a remarkable delay in SB reprotonation [154]. In contrast to the cases of BR or LR, the proton donating function of GPR was not lost by the substitution of Glu108GPR with BR and LR-like aspartate or even ESR-like lysine (data unpublished). Therefore, the distinct mechanisms of CP proton translocation via their proton donors and the specificity of the respective proton donors in the three types of H+ -pumping rhodopsins may originate from the difference in the environment around each proton donor in the CP channel.
