**1. Introduction**

Optical control of biological reactions is one of the most recently studied fields of research because light facilitates highly spatial and temporal manipulation. In particular, optogenetics, that is, the specific and noninvasive control of biological activities such as neural activities by light stimulus of photoreceptor proteins heterogeneously expressed in targeted neurons or other related cells, has a significant impact in the field of neuroscience [1–8] and has attracted the interest of myriad researchers in the life sciences. Over the past 15 years since the first report on optogenetics in 2005 [1], the development of tools for this interesting technique has been rapidly progressing [9–14]. Recently, various types of photosensitive proteins have been employed for optogenetics [15–17]. Nevertheless, retinal-based proteins found in microbes (referred to as microbial rhodopsins), which were first applied to optogenetics, are still overriding toolkits [18, 19].

Microbial rhodopsins (also termed type-I rhodopsins) are seven transmembrane αhelical proteins that bind to the retinal chromophore, similar to animal rhodopsins (also termed type-II rhodopsins) [20]. A distinctive property of animal rhodopsins is the difference in their chromophore configurations; retinals in microbial and animal rhodopsins adopt all-*trans* and 11-*cis* forms in the dark state, respectively. In addition, by all-*trans*-to-13-*cis* isomerization of the retinal with illumination, microbial rhodopsins undergo a linear cyclic photoreaction called photocycle, in contrast to animal rhodopsins, whose retinals are isolated from the protein moiety during their photoreaction processes. Their functions are also different; in addition to photo-sensing functions of animal rhodopsins, microbial rhodopsins also act as light dependent-ion transporters that can carry various types of ions such as H+ , Na<sup>+</sup> , and Cl [21–24].

Microbial rhodopsins are classified into two categories of ion carriers. One is a lightgated ion channel, and the other is a light-driven ion pump. The former group includes channelrhodopsins (ChRs) [8, 25–27] and anion channelrhodopsins (ACRs) [28–30], which are the principal tools for optogenetics. Upon illumination, ChRs become permeable to various monovalent or divalent cations, such as H+ , K+ , Na+ , and Ca2+ [8, 25–27]. Therefore, in nerve cells expressing ChRs, the influx of Na+ induced by light activation of ChRs causes depolarization in these cells, leading to neural activation [1–8, 25–27]. Conversely, light activation of ACRs, which act as anion-selective channels, can drive the hyperpolarization of ACR-expressing cells to suppress neural activity [28, 31]. The ion pump group includes light-driven outward H+ - [32, 33], Na+ - [34], and inward Cl pumps [35–38]. As these proteins can generate negative membrane potential in their incorporated cells by illumination, they can be utilized as neural silencers similar to ACRs [39, 40]. Microbial rhodopsins, as ion channels or pumps, can lead to changes in membrane potential by absorption of a photon without going through complicated reactions. This simple light-activated machinery makes them more easily applicable to optogenetics, together with repeatable properties through their photocycle.

Among the three types of ion-pumping rhodopsins, proton-pumping rhodopsins have a distinct feature from the other two. Proton translocation across the cell membrane induced by light activation of these pigments is accompanied by a change in intracellular pH. Hence, these proteins have the potential for various applications, for example, photoinduced pH control in cells or all sorts of organelles, as well as their use as neural silencers. To date, genes encoding H+ -pumping rhodopsins have been identified from the genomes of many microorganisms, irrespective of species [41], which enables us to gain the most plentiful genetic information from the database of the microbial rhodopsin family. Therefore, these types of rhodopsins may be applicable for exploring better candidates for optogenetics in various respects, such as the strength of neural inhibition, spectral properties (maximum absorption wavelength for activation), and kinetics.

Chow et al. screened efficient neuronal silencing rhodopsins and showed that the magnitude of photocurrents evoked by the activation of H+ -pump-type rhodopsins

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

was on average higher than those evoked by the activation of inward Cl-pump halorhodopsins (HRs) [39]. Moreover, the rates of activation upon light irradiation and recovery from inactivation after light cessation tended to be faster, as observed for archaerhodopsin-3 from *Halorubrum sodomense* (aR-3 or Arch), which is currently the most powerful H<sup>+</sup> -pumping tool for neural suppression, unlike HRs that retain long-lasting inactive states [39]. Based on these observations, H<sup>+</sup> -pump rhodopsins are considered more effective for the light-induced inhibition of neurons. Thus, these experimental facts for the practical use of H+ -pumping rhodopsins have been steadily amassed; however, the utility of H+ -pumping rhodopsins for optogenetics has not been completely evaluated from the molecular viewpoint. Therefore, an overview of the molecular mechanism of various H<sup>+</sup> -pumping rhodopsins, including newly found H+ -pumps, may be useful for further development and rational design of optogenetic instruments. Here, we describe the functional mechanism of H<sup>+</sup> -pumping rhodopsins, particularly highlighting the aspect of photochemistry and the accompanying proton movement, with their future prospects for optogenetic applications.

### **2. H<sup>+</sup> -pumping rhodopsins from various microbial species**

### **2.1 H+ -pumps in archaebacteria**

Among all microbial rhodopsins, the first H+ -pumping rhodopsin reported was bacteriorhodopsin (BR), which was discovered in *Halobacterium salinarum* living in salt lakes or salterns in 1971 [42]. Haloarchaea, including those described above, can survive even in extremely salty environments with low oxygen concentrations using BR-based phototrophy, which is accomplished by ATP synthesis driven through a proton gradient produced with outward proton translocation across the cell membrane. Haloarchaeal plasma membranes contain deeply purplish patches (referred to as purple membranes), in which BR forms highly dense assemblies in the form of a two-dimensional hexagonal lattice. The high BR expression in native membranes, along with its highly stable property, facilitated biochemical and biophysical investigations of this protein by various approaches, including spectroscopic and structural methods [32, 33, 43–48]. Thus, BR is the most well-studied H+ -pump.

Following the discovery of BR, the second H<sup>+</sup> -pump identified was archaerhodopsin (aR). Two homologous proteins, archaerhodopsin-1 and -2 (aR-1 and aR-2), were simultaneously identified from *Halobacterium* sp. aus-1 and aus-2 isolated from a lake in Western Australia by Mukohata et al. [49]. Several aR homologous proteins, including aR-3 described above, have been discovered in different haloarchaeal species [50–53]. In addition, Mukohata et al. successively identified two other H<sup>+</sup> -pump-like proteins belonging to a different clade from BR and aR: cruxrhodopsin-1 (cR-1) from *Haloarcula argentinensis* [54] and deltarhodopsin-1 (dR-1) from *Haloterrigena* sp. arg-4 [50]. Several homologs of these H+ -pumps have also been identified in other species [55–57]. aRs, cRs, and dRs are very similar H<sup>+</sup> pumps to BR; however, they are classified as apparently different tribes [50].

### **2.2 Eubacterial H+ -pumps**

The history of microbial rhodopsin research has been confined to the archaebacterial world for about three decades since the first discovery of BR. However, since the 2000s, rapid technical advances in metagenomics have led to the discovery of unknown

microbial H+ -pumping rhodopsins from various eubacteria [58, 59]. A representative example is proteorhodopsin (PR) from marine bacteria [60, 61].

In 2000, PR was first identified in the genome of uncultivated marine γproteobacteria, which is a member of the SAR86 clade, from a sea sample collected from Monterey Bay in California [62]. Thus, the nomenclature of this protein, i.e., "proteo-," originates from the name of the hosting bacterium. Sequencing of a bacterial artificial chromosome vector into which a fragmented DNA extracted from samples was cloned revealed the presence of a gene encoding rhodopsin-like protein (EBAC31A08) [62]. Furthermore, after transformation by this gene and successive induction of protein expression with exogenous retinal in *Escherichia coli*, acidification in suspension containing these PR-expressing cells was caused by illumination, indicating that PR can work as an outward light-driven BR-like H+ -pump in the *E. coli* membrane [62]. After the first discovery of PR, further surveys demonstrated the existence of genes encoding novel PRs in not only γ-proteobacteria but also α-proteobacteria containing ubiquitous marine clades such as the SAR11 group [63], β-proteobacteria [64], and Flavobacteria [65, 66]. In addition, genes encoding numerous PR variants (>several hundreds or thousands of variants) have been identified in widespread oceans [67–72]. Nowadays, most marine bacterioplankton living in the photic zone are assumed to hold PR genes [41]. PR can be classified into two groups depending on their absorption maxima (λmax): green-absorbing PR (GPR), whose λmax is approximately 525 nm, and blue-absorbing PR (BPR) with a λmax of ca. 490 nm [67, 69, 73–75]. The difference between these two groups is probably associated with the adaptation to the environments that the PR-retaining bacteria inhabit; most bacteria that are distributed at the surface of the sea and have access to available green light have GPR to obtain energy produced effectively using this wavelength of light, while bacteria at the depth of the sea water that exclusively have access to available blue light contain BPR [67, 74, 75].

PR-related proteins were also discovered from non-marine bacteria present in various environments, such as freshwater [76], high mountains [77], hot springs [78], and permafrost [79]. For example, a PR-like protein identified from actinobacteria living in freshwater is called actinorhodopsin (ActR) because it is classified into a phylogenetically different clade from PR [76]. A halophilic eubacterium *Salinibacter ruber* also contains a PR-like H<sup>+</sup> -pumping protein called xanthorhodopsin (XR) [80]. XR binds to the second chromophore, carotenoid salinixanthin, which acts as a lightharvesting antenna, expanding the spectral range for light activation of this protein because the energy obtained by light absorption of salinixanthin can be transferred to the retinal to induce isomerization [80, 81]. Another PR-like H<sup>+</sup> -pump with binding ability to salinixanthin, similar to XR [82], was discovered from the cyanobacterium *Gloeobacter violaceus* and called *Gloeobacter* rhodopsin (GR) [83]. Furthermore, a new type of H+ -pump with a unique feature (described later) was discovered from a nonmarine gram-positive bacterium *Exiguobacterium sibiricum* present in Siberian permafrost samples, which was named *Exiguobacterium sibiricum* rhodopsin (ESR) [79]. Thus, PR-like eubacterial H+ -pumping rhodopsins have been found in various archaea and bacteria [84, 85] and even in eukaryotic marine protists [86], which seems to have been achieved by lateral gene transfer [84].

### **2.3 Two types of H<sup>+</sup> -pumps from lower eukaryotes**

In 1999, the presence of a gene encoding eukaryotic microbial rhodopsin (*nop-1*) was first found in the eukaryotic filamentous fungus *Neurospora crassa* [87]. This
