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


### **Figure 2.**

*Amino-acid alignment of various microbial H<sup>+</sup> -pumping rhodopsins. Analysis was performed using a multiple sequence alignment program (CLUSTALW). The numbers shown in the top row represent the numbering of amino acid residues in BR. The dotted line represents the missing residues in the determined structure. The amino acid residues with maximum homological numbers at each position are marked with a black or gray background depending on their numbers: The monochrome tone becomes darker as the number of homological residues increases. Notes: cR-2, cR from Haloarcula sp. arg-2; cR-3, cR from Haloarcula vallismortis; dR-2, dR from Haloterrigena turkmenica JCM9743; dR-3, dR from Haloterrigena thermotolerans; GPR, γ-proteobacterium (EBAC31A08) GPR; BPR, γ-proteobacterium (Hot75m4) BPR; ActR, RpActR.*

The p*K*<sup>a</sup> value of PRC in the H+ -releasing M-state (6) [100] divides the timing of proton release into two patterns: at pH values above 6, a proton is initially released from PRC to the EC bulk during the L–M transition and the resultant deprotonated PRC receives a proton from the protonated Asp85BR upon O-decay. In contrast, at pH values below 6, the first such proton release from PRC upon M-rise does not occur, and a proton on PRC is released late upon O-decay [101].

Two threonine residues, Thr89BR and Thr46BR, are also important, although these residues do not belong to the series of proton transfer events due to nonionizable residues. Thr89BR is within the active center and includes PSB, Asp85BR, and some water molecules [102], where this residue forms a hydrogen bond with Asp85BR [103], indirectly contributing to the initial proton transfer from PSB to Asp85BR during Mformation [102, 103]. In contrast, Thr46BR forms an interhelical hydrogen bond with Asp96BR in the CP region, which is associated with the regulation of p*K*<sup>a</sup> in Asp96BR in the unphotolyzed state [104].

### **3.2 Common and different points on the amino-acid sequences among varying H+ -pumps**

In most outward H+ -pumping microbial rhodopsins identified to date, the residues corresponding to three main groups (PSB [Lys216BR as the retinal binding site], Asp85BR, and Asp96BR) described above are highly conserved. By checking their presence, we can therefore forecast whether each protein in the microbial rhodopsin family acts as an H+ -pump like BR. **Figure 2** shows a comparison between important amino acid residues for proton transport among representative H+ -pumping rhodopsins. As shown in this figure, almost all primal residues relevant to proton transport in archaealtype H+ -pumps agree with the residues corresponding to BR. Similarly, both fungal and algal H+ -pumps from eukaryotes retain the residues corresponding to Asp85BR and Asp96BR; however, a difference exists in the components of PRC in BR. In both types of eukaryotic H+ -pumps, the residue corresponding to Glu194BR of two EC glutamates in PRC is replaced by glycine, whereas another residue corresponding to Glu204BR is conserved. In contrast, in the eubacterial H+ -pump, the residues corresponding to Asp96BR are substituted by conservative carboxylate glutamic acid, although there are several exceptions. Another significant aspartate corresponding to Asp85BR is perfectly conserved, similar to other types of H+ -pumps. Furthermore, these H+ -pumps lack both glutamic acids in the components of PRC: Glu194BR and Glu204BR. Thus, a comparison of the amino acid sequences among various H+ -pumping rhodopsins can reveal the superconservation of the proton acceptor (Asp85BR) and the diversity of the proton donor (Asp96BR) and the residues in the EC proton releasing pathway. These differences could lead to different methods of proton transfer among varying H+ -pumps.

### **3.3 Photocycles of other H+ -pumping rhodopsins than BR**

During a single photocycle induced by the absorption of one photon, ion-pump-type rhodopsins can transport ions as substrates. The number of photocycle turnover under illumination, therefore, affects the amount of ions transported by these proteins, in other words, the ion-pumping activity of these rhodopsins. In general, the turnover rate of the photocycle in ion-pumping rhodopsins tends to be relatively higher than those of photosensor-type rhodopsins to transport numerous ions per illumination. The speed of their photocycle completion can be used to analyze the H+ -pump, in addition to actually measuring H+ -pumping activity that is usually examined by measuring the photoinduced pH change in a suspension of cells expressing these rhodopsins. Furthermore, the identification of photointermediates during the photocycle of respective rhodopsins and the estimation of their rise/decay kinetics together with the measurement of transient proton transfer during their photocycles enable us to understand the timing of proton movement. Thus, detailed investigations of the photocycles are important for understanding the H+ -pumping mechanism.

Among the H<sup>+</sup> -pumping rhodopsins identified so far, the next well-characterized proton pump following BR is GPR. In many studies, the first identified PR variant (EBAC31A08) was employed as a sample. As soon as GPR was discovered in 2000, various spectroscopic approaches such as static and time-resolved transient

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

UV–visible, FTIR, and FT-Raman spectroscopies were applied to characterize the photochemistry of this protein, as previously performed for the research of BR [105– 110]. These experimental results revealed that the photocycle of GPR was similar to that of BR but also concomitantly contained several differences. Using the same kinetically analytical method as previously applied to the transient absorbance data of BR, where the possibilities of parallel or branch models were also considered [111], Váró et al. determined the photocycles of GPR at acidic and alkaline pH values [108, 109]. Their proposed photocycle at alkaline pH (9.5) is in accordance with the following scheme: GPR ! K\$M1 ! M2\$N\$GPR'(O) ! GPR [108]. As shown in the above scheme, one of the apparent differences from the BR photocycle is the absence of L after K, which is thought to be probably due to kinetic reasons. A remarkably retarded (ca. 10-100-fold slower) decay of K compared with that of BR was observed in GPR [105]. Because of such slow K-decay, low-temperature Raman spectroscopic data presented by Fujisawa et al. demonstrated that the chromophore structure in GPR in the K state is less distorted compared to that of BR in the same state and is rather close to that of L in BR, which possess a more relaxed chromophore structure [112]. Therefore, the formation of a longer stable K state may obscure the appearance of L in the GPR along with the fast formation of the following M-state. Another difference from BR can be observed in the spectral characteristics of the latter photoproducts, N and GPR'(O). The N-intermediate in PR was red-shifted with 13-*cis* chromophore retinal [105, 108] and resembled O in BR with respect to the absorption maxima. In addition, the GPR' intermediate had all-*trans* retinal chromophores similar to O in BR; however, its λmax was very close to that of the original pigment. Friedrich et al. also determined the photocycle of GPR under both acidic (pH 5) and alkaline (pH 10) conditions based on a global fitting analysis (sequential irreversible model) for flash photolysis data [106]. In the latter half of the photocycle scheme proposed by them, after an equilibrium of M and a red-shifted O (λmax = 580 nm) was produced, an equilibrium of N with a spectral property similar to that of the original pigment (λmax = 530 nm) and O appeared [106]. Hence, if it is assumed that O and N in their scheme agree with N and GPR' in Váró's scheme, respectively, both schemes are compatible. The rate of photocycle turnover in GPR was fast (<several hundreds of milliseconds), although it was somewhat slower than that of BR (<several tens of milliseconds). In contrast, the photocycle of another type of PR, BPR, was slower by an order of magnitude than that of GPR [113]. The possibility of using BPR as a photosensor has been advocated, although its physiological role is still debated [113].

The photocycles of other eubacterial H+ -pumping rhodopsins, including XR, GR, ESR, and ActR, were also investigated by time-resolved absorption spectroscopy [80, 83, 114–116]. Their photocycles go through the K, L, M, N, and O states, similar to BR or GPR. For many eubacterial H+ -pumps including GPR, structural information obtained by multiple approaches such as X-ray crystallography, NMR, and atomic force microscopy has also been reported [117–123], providing structural insights into their photochemistry.

Recent genome analysis revealed that numerous eukaryotic fungi possess rhodopsinlike protein-encoding genes (RDs) and opsin-related genes (ORPs) [124]. Nevertheless, unlike archaeal or bacterial H+ -pumping rhodopsins, reports on the photochemistry of eukaryotic H+ -pumps are extremely limited because the protein expression in *Pichia pastoris* has been established only for a few fungal rhodopsins such as NR and LR. Meanwhile, several studies on the photochemical characterization of LR and its analogous protein PhaeoRD1 using visible and infrared spectroscopic techniques have been published [91, 92, 125–128]. These reports revealed that their photocycles include the K, L, M, N, and O states, similar to the BR photocycle [91, 92].

For two algal H+ -pumps, ARI and ARII, the establishment of a large-scale sample preparation method using a unique *Escherichia coli* cell-free membrane-protein production system developed by Shimono et al. [129] allowed the detailed elucidation of the spectroscopic and structural features of these proteins [96, 130–133]. Through global fitting analysis for time-dependent absorption changes based on a sequential irreversible model, we determined that the photocycles for ARI and ARII at nearneutral pH values can be represented by ARI ! K\$L\$M\$N\$O ! ARI' ! ARI and ARII!K ! L\$M\$N\$O ! ARII' ! ARII, respectively, which are very similar to the BR photocycle [130, 131]. However, the formation of a long M-N-O quasi-equilibrium was observed in both the photocycles of both AR proteins [130, 131], which is characteristic of these ARs. This indicates the presence of pronounced reverse reactions between M, N, and O in the photocycles of ARI and ARII. Although similar N–M or O–N back reactions also exist in BR, the rates of these reactions in BR are not significantly higher than those of ARI and ARII. The existence of prompt back reactions could hamper the fast turnover of the photocycle in these rhodopsins, thereby reducing H+ -pumping efficiency. However, a significantly faster O-rise and the irreversibility of the transitions from O to ARII' (a precursor of ARII) and from ARII' to the original state were observed during the photocycle of ARII [130]. Owing to these kinetic properties, the overall photocycle of ARII is a forward reaction, which may result in a turnover rate (<�100 ms at neutral pH) that is comparable to that of BR [130]. In contrast, the photocycle turnover of ARI was approximately 10-fold slower than that of ARII [131]. This may be attributed to slower decay of O and ARI' in the second half of the photocycle in ARI compared to the decay of O and ARII' in the photocycle of ARII.
