**3.6 Existence of two substates in the latter photoproducts of the photocycle and the chemical and structural events occurring during the transition between them**

Among the three photointermediates M, N, and O produced in the latter half of the photocycle in H<sup>+</sup> -pumping rhodopsins, two spectrally silent substates are known for each photoproduct [33, 132]. Because the transitions between these substates occur without apparent spectral changes, they are usually observed by kinetic analysis for transient absorbance changes measured using various spectroscopic techniques. Three critical events for proton translocation occur during these silent transitions. As is known in BR, the first crucial event was observed upon the transition between two successive M-states, M1 and M2, which is accompanied by the accessibility switch of SB from the EC side to the CP side. This switching is important for unidirectional proton transport because it causes the conversion of the direction of proton movement from toward EC at M to toward CP at N.

The second event occurs during the N1-to-N2 transition, where the accessibility of the proton donor changes. In BR, the proton donor Asp96BR connects to the SB but not the CP bulk during the M–N transition, thus hampering the misdirected transfer of a proton of Asp96BR toward the CP solvent. Then, the connection of Asp96BR to SB is switched toward the CP side upon the N1–N2 transition, facilitating the reprotonation

of Asp96BR from the CP surface [33, 45]. Although the detailed mechanism of this accessibility switch upon N1–N2 transition remains incompletely understood, even in the most well-known BR, a previous computational study by Wang et al. proposed a model in which the further opening of the proton uptake pathway in the CP channel, which remains closed even in the M-state with the opening of the F-helix by the presence of a hydrophobic barrier composed of Phe42BR and multiple other hydrophobic residues, is triggered by the deprotonation of Asp96BR during the M–N transition, leading to the connection of Asp96BR to the CP aqueous space [155]. In contrast, for the algal H+ -pump ARII, it was presumed that the change in the unique interhelical interaction between Asp92ARII and Cys218ARII located in the CP domain acts as a switch for opening the gate of the CP channel for H+ -uptake [133].

In contrast to M and N, the molecular events in the O-state have not been completely examined because the stable trapping of O produced in the latter stages of the photocycle is difficult. In the early stages of studies on BR, Haupts et al. hypothesized that during the N–O transition, the reisomerization of the retinal from the 13-*cis* (15-*anti* PSB) to all-*trans* (15-*syn* PSB) form is followed by the switching of the N-H bond of PSB from the CP (15-*syn* PSB) to the EC (15-*anti* PSB) side, the so-called isomerization/switch/transfer (IST) model [156]. In contrast, the results of MD simulation performed by Wang et al. supported the opposite model (SIT model) as a more plausible scheme: the isomerization of the retinal from 13-*cis* (15-*syn* PSB) to all-*trans* (15-*anti* PSB) is preceded by the switching of PSB from the 15-*anti* to 15-*syn* forms [157]. If the scheme corresponds to the latter model, another substate with a 13-*cis* chromophore should be formed after the switching of PSB during the N2-O transition with the thermal reisomerization of retinal. Thus, we attempted to detect the presence of further substates. In general, the existence of the quasi-equilibrium among M, N, and O states described above makes it difficult to observe O. However, in the algal H+ pump ARII under acidic conditions (pH < 5.5), N did not accumulate during the photocycle due to the presence of a rapid back reaction between M and N and the acceleration of proton uptake upon the following N–O transition under these conditions, resulting in notable observation of O [132]. Through kinetic analysis of timeresolved absorbance changes under these conditions, we succeeded in detecting two spectral analogous O-intermediates (O1 and O2) [132]. As the O1–O2 transition was accompanied by a faint but obvious red-shift of the absorption maximum, we assumed that the 13-*cis*-to-all-*trans* retinal isomerization occurs during the O1–O2 transition after the switching of PSB upon the N2–O1 transition based on the model proposed by Wang et al. [157]: O1 is a precursor before the formation of O (O2) with a twisted all-*trans* chromophore retinal. In previous studies on BR, it was reported that the steric contact of Lue93BR with the 13-methyl group of retinal is significant for facilitated retinal reisomerization during this transition [158]. The residue corresponding to this leucine is almost completely conserved among all microbial rhodopsins (see **Figure 2**), implying that the mechanism described above is common in the microbial rhodopsin family.

### **3.7 Role of PRC formed on the EC surface**

As described above, PRC located on the EC surface is not necessarily indispensable for proton pumping because of the presence of a PRC-deficient type (eubacterial or eukaryotic) H+ -pumping rhodopsins, although PRC alters the timing of proton release during the photocycle. The replacement of either Glu194BR, Glu204BR, or both by nonionizable residues, however, caused a delay in O-decay with a late proton release

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

from the protonated Asp85BR toward the EC surface as well as the absence of the initial proton release upon the L–M transition. In addition, when the residues corresponding to three of PRC-constituting residues (Ser193BR, Glu194BR, and Thr205BR) in a sensory-type rhodopsin from *Natronomonas pharaonis* (NpSRII) were replaced by the same residues as BR, the lifetime of O in this triple NpSRII mutant became approximately 20-fold shorter than that of the wild type [159]. Hence, the presence of PRC on the EC surface may be involved in not only the early proton release toward the EC aqueous phase during the photocycle but also in the formation of a hydrophilic proton conductive pathway in the EC channel, which contributes to efficient proton translocation. In contrast, as observed in the X-ray crystal structure of ESR, there may be a cavity for the proton releasing pathway that already connects to the EC bulk solvent at the resting state in eubacterial H<sup>+</sup> -pumping rhodopsins without PRC. Thus, in the EC domain of these H<sup>+</sup> -pumping rhodopsins, a hydrophilic pathway may be formed in a different manner from archaeal H+ -pumps, participating in facilitated proton movement on the EC side.
