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

rhodopsin-like protein encoded by *nop-1* is called *Neurospora* rhodopsin (NR). The amino acid sequence of NR contained the requisite corresponding residues for proton pumping of BR; however, a previous photochemistry study using recombinant NR proteins heterogeneously expressed in the methylotrophic yeast *Pichia pastoris* revealed that NR showed a slower photocycle that is close to sensor-type rhodopsins [88]. Therefore, it is speculated that NR is physiologically associated with carotenoid biosynthesis regulation by functioning as a photosensor rather than a H+ -pump [89, 90], although its exact physiological role remains unknown. Later, other NRrelated fungal opsin genes were discovered in a different fungal species, *Leptosphaeria maculans*, which is the fungal agent of blackleg in canola [91]. This opsin-coded protein is termed *Leptosphaeria* rhodopsin (LR or Mac). Through its characterization using proteins prepared by heterogeneous expression in yeast (*Pichia pastoris*) similar to NR, it was demonstrated that LR acts as a BR-like outward H<sup>+</sup> -pump with a fast photocycle, unlike NR [91]. Furthermore, through advanced genomic analyses, new fungal rhodopsins that are classified into a third subgroup were identified. The fungal wheat pathogen *Phaeosphaeria nodorum* possesses two rhodopsin-like proteinencoding genes [92]. These fungal rhodopsins are called *Phaeosphaeria* rhodopsin 1 (PhaeoRD1) and *Phaeosphaeria* rhodopsin 2 (PhaeoRD2). PhaeoRD1 is an analogous protein to LR, whereas PhaeoRD2 is a member of the third group. Considering its coexistence with other rhodopsin forms from the same species, PhaeoRD2 is regarded as an auxiliary protein [92]. Characterization of these fungal rhodopsins heterogeneously expressed in *P. pastoris* suggested that both pigments exhibit fast photocycles that are characteristic of H+ -pump-type rhodopsins [92].

*Phylogenetic tree of microbial rhodopsins. RpActR represents ActR from actinobacterium Rhodoluna planktonica strain MWH-Dar1.*

*Acetabularia* rhodopsin (AR) is another eukaryotic H<sup>+</sup> -pump found in the giant unicellular marine green alga *Acetabularia acetabulum* [93]. *Acetabularia acetabulum*, which is also known as the "Mermaid's Wineglass", is an extremely interesting organism in terms of morphology because this unicell exhibits a unique complex life cycle comprising several distinct developmental phases [94]. In 2004, Mandoli et al. first reported the cDNA sequence of a fragmented possible opsin-encoding gene (*aop*) from juvenile *Acetabularia*. Subsequently, Hegemann et al. succeeded in cloning fulllength opsin cDNA from this alga [93]. They heterogeneously expressed AR proteins in the membrane of *Xenopus laevis* oocytes and characterized the electrophysiological properties of this protein. Through a series of experiments, they demonstrated that AR is an outward light-driven H<sup>+</sup> pump [93]. Moreover, Jung et al. successively recloned two opsin genes from juvenile *Acetabularia*, which slightly differed from the gene cloned by Hegemann et al. The two AR homologs identified by them were named *Acetabularia* rhodopsin I and II (ARI and ARII, also abbreviated as Ace1 and Ace2, respectively) [95, 96]. Thus, two types of H<sup>+</sup> -pumps from eukaryotic microorganisms are currently known: fungal and algal H+ -pumping rhodopsins (**Figure 1**).

### **3. Proton translocation mechanism of microbial H<sup>+</sup> -pumping rhodopsins: from the photochemical and proton transfer viewpoints**

### **3.1 Proton transport of BR: a typical model of H<sup>+</sup> -pumping rhodopsins**

When the molecular mechanism of microbial H<sup>+</sup> -pumping rhodopsins is considered, the scenario of proton transportation in BR is often used as a prototype. Detailed descriptions of the H<sup>+</sup> -pumping mechanism of BR from various aspects can be found in excellent previously published reviews (refer to relevant refs. [32, 33, 43-48]). We present only a brief outline here.

The photocycle of BR is initiated by photoisomerization of the retinal from all*trans* to 13-*cis* upon formation of the K-intermediate. Then, during the transition between four sequentially formed photoproducts, L, M, N, and O intermediates, stepwise proton transfer reactions occur between amino acid residues buried within the protein or aqueous phases on both the cytoplasmic (CP) and extracellular (EC) sides. In these processes, three main groups play an essential role in proton transport. One is a part of the retinal Schiff base (SB), which represents a linkage with a specific lysine residue located at the center of the seventh helix of the protein (G-helix) (Lys216BR, **Figure 2**). This portion is usually protonated in the unphotolyzed state (protonated retinal Schiff base, PSB). The other groups are two aspartic acid residues, Asp85BR and Asp96BR, located in the EC and CP domains, respectively, on the C-helix. Asp85BR facilitates the first step of proton translocation upon the L–M transition as a proton acceptor from PSB, whereas Asp96BR works as a proton donor to deprotonated SB during M–N transition and is sequentially involved in proton uptake from the CP bulk upon N–O transition accompanied by 13-*cis*-to-all-*trans* retinal reisomerization. Both Asp85BR and Asp96BR are required for efficient proton pumps because substitutions of these residues with nonionizable residues abolished or significantly decreased H+ -pumping capability [97].

A proton releasing complex (PRC) comprising several internal H2O and various residues on the EC surface such as Tyr57BR, Arg82BR, Tyr83BR, Ser193BR, Glu194BR, Glu204BR, and Thr205BR also participates in the proton transfer reaction of BR [98, 99], although it is not always an indispensable component for proton pumping.
