A single mutation converts bacterial Na<sup>+</sup>‐transporting rhodopsin into an H<sup>+</sup> transporter

Mamedov, Mahir D.; Mamedov, Adalyat M.; Bertsova, Yulia V.; Bogachev, Alexander V.

doi:10.1002/1873-3468.12324

Cited by 16 publications

(11 citation statements)

References 30 publications

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“…In reality, KR2 pumps sodium ion exclusively under physiological conditions in which the concentration of the sodium ion is much greater than that of protons. The fact that Q123D/E only pumps protons is consistent with the competitive uptake model, 83 where Q123 contributes to efficient sodium uptake (Figure 5B, left). 64,95 A comparison of the permeability of channels, transporters, and flagella motor complexes to sodium ions and protons has been reported.…”

Section: Photocycle Dynamics Of Light-driven Sodium-pumping Rhodopsinssupporting

confidence: 82%

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport

Kandori¹,

Inoue

Tsunoda

2018

Chem. Rev.

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Ion pumps perform active transport of ions by using energy. The active transport mechanism can be illustrated by the Panama Canal model, which considers two gates and a gain in energy. The Panama Canal model is consistent with the alternating access model that is used to describe active transport, in which the substrate ion is bound, energized, and released. It was generally accepted that energization occurs only for an ion-bound protein but not for an ion-unbound protein. Light-driven proton and chloride pumps, two of the best studied pumps, are represented by the Panama Canal model. In this case, light absorption takes place for the bound state of ions (proton and chloride ions) in the active center (protonated Schiff base of the retinal chromophore). In contrast, a recently discovered light-driven sodium pump, Krokinobacter eikastus rhodopsin 2 (KR2), is a unique active transporter that does not bind the transport substrate, the sodium ion, in its resting state. The molecular architecture and photoreaction cycle of the light-driven sodium pump are very similar to those of proton and chloride pumps, although sodium ions are actively transported without initial binding. Sodium uptake is a diffusive process, but the presence of two gates allows the unidirectional transport of sodium ions. In this sense, the light-driven sodium pump is also represented by a modified Panama Canal model. Current understanding of the light-driven sodium pump is reviewed.

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Section: Photocycle Dynamics Of Light-driven Sodium-pumping Rhodopsinssupporting

confidence: 82%

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport

Kandori¹,

Inoue

Tsunoda

2018

Chem. Rev.

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“…oxidase [28,29], and Na + -proteorhodopsins [59,60]. Consistent with the above explanation for the effect of HQNO, we showed that in the presence of HQNO, the Na + /H + antiporter monensin (dissipating transmembrane [Na + ] and [H + ] gradients) abolished the HQNO-induced stimulation of the motility rate (Figure 6c).…”

Section: Resultssupporting

confidence: 87%

“…As a result, the activity of the primary Na + pump increases, followed by an increase in ∆pNa with the simultaneous dissipation of the electric potential on the membrane. The stimulation effect of protonophore uncouplers on Na + pumping has been described for a number of ∆s-generating proteins [52], among which are Na + -pumping NADH-quinone oxidoreductases [53], Na + -ATPases [54][55][56][57][58], recently described Na + -pumping cytochrome oxidase [28,29], and Na + -proteorhodopsins [59,60]. Consistent with the above explanation for the effect of HQNO, we showed that in the presence of HQNO, the Na + /H + antiporter monensin (dissipating transmembrane [Na + ] and [H + ] gradients) abolished the HQNO-induced stimulation of the motility rate (Figure 6c).…”

Section: Resultsmentioning

confidence: 99%

Sodium Energetic Cycle in the Natronophilic Bacterium Thioalkalivibrio versutus

Muntyan

Viryasov

Sorokin

et al. 2022

IJMS

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As inhabitants of soda lakes, Thioalkalivibrio versutus are halo- and alkaliphilic bacteria that have previously been shown to respire with the first demonstrated Na+-translocating cytochrome-c oxidase (CO). The enzyme generates a sodium-motive force (Δs) as high as −270 mV across the bacterial plasma membrane. However, in these bacteria, operation of the possible Δs consumers has not been proven. We obtained motile cells and used them to study the supposed Na+ energetic cycle in these bacteria. The resulting motility was activated in the presence of the protonophore 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), in line with the same effect on cell respiration, and was fully blocked by amiloride—an inhibitor of Na+-motive flagella. In immotile starving bacteria, ascorbate triggered CO-mediated respiration and motility, both showing the same dependence on sodium concentration. We concluded that, in T. versutus, Na+-translocating CO and Na+-motive flagella operate in the Na+ energetic cycle mode. Our research may shed light on the energetic reason for how these bacteria are confined to a narrow chemocline zone and thrive in the extreme conditions of soda lakes.

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“…10 Understanding the key residues involving in the ions transports in microbial rhodopsins permits the possibility to manipulate the pumping activities. 38,39 Site directed mutagenesis was applied to convert pumping activity. By converting the proton accepter in BR, D85T pumps Clinwardly similar to HR.…”

Section: Retinal Proteinsmentioning

confidence: 99%

“…The advantage of cells hyperpolarization using sodium ion instead of proton is the capability to manipulate the cell membrane potential without affecting pH of the cells. 170,171 After the discovery, numerous studies based on time-resolved visible 2,48 , FTIR 3,52,54,172 , Raman 4 , NMR spectroscopies 5,173 , X-ray crystallography 16,17,50 , and site directed mutagenesis 1,16,17,38,40 have been performed to elucidate the molecular mechanism and ion transport pathway of the protein. Crystal structures of the wild type and important mutants in the dark state 16,17,50 have been reported on the PDB database.…”

Section: Transport Assaymentioning

confidence: 99%

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

Jakdetchai¹

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KR2 is a light-driven sodium ion pump found in marine flavobacterium Krokinobacter Eikastus. The protein belongs to the microbial rhodopsin family, which is characterized by seven transmembrane helices and a retinal cofactor covalently bound to a conserved lysine residue through a Schiff base linkage. Specific features of KR2 and other sodium pumping rhodopsins are the NDQ motif, the N-terminal helix capping the protein at the extracellular side, and the sodium ion bound at the protomer interface in the pentameric structure. The ability to pump sodium ions was a surprising discovery since the positive charge at the Schiff base was long thought to hinder the transport of non-proton cations and the Grotthuss mechanism could not be applied to explain the Na+ transport. The photocycle of KR2 revealed by flashed photolysis and ultrafast femtosecond absorption spectroscopy consists of consecutive intermediates, named K, L, M, and O. Here, DNP-enhanced ssNMR was used to analyze various aspects of these intermediate states. The K/L-state can be generated and trapped by in-situ illumination inside the magnet at 110 K. The trapping of L-state together with the K-state at this temperature is unexpected as this usually leads to the trapping of only K-state in bacteriorhodopsin (BR), proteorhodopsin (PR), and channelrhodopsin 2 (ChR2). This observation suggests a lower energy barrier between K- and L-state in KR2. For the O-state, the intermediate was generated by illuminating outside the magnet, followed by rapid freezing in liquid nitrogen and transfer to the magnet. Based on these procedures, the retinal conformation, and the electrostatic environment at the Schiff base in KR2 dark, K-, L- and O-intermediates were probed using 13C-labeled retinals bound to 15N-labeled KR2 by both 1D and 2D magic angle spinning (MAS) NMR experiments. The obtained data show an all-trans retinal conformation with the distortion of 150° at H-C14-C15-H in the dark state whereas the retinal has a 13-cis, 15-anti conformation in the K- and L-state after light activation. Differences between K- and L-intermediates were observed. The retinal chemical shifts of the K-state show a large deviation from the model compound behavior between the middle and end part of the polyene chain. In the L-state, these differences are much less pronounced. These observations indicate that the light energy stored in the K-state dissipates into the protein in the subsequent photointermediate states. Furthermore, an additional shielding observed for C14 in L-state indicates the slight rotation toward a more compact 13-cis, 15-syn conformation. The distortion of the H-C14-C15-H angle in the L-state (136°) is larger than in the dark state. This twist of the retinal in the L-state would play an important role in lowering the pKa of the Schiff base, which is a prerequisite for the proton transfer from the Schiff base to the proton acceptor (D116). The electrostatic environments at the Schiff base in K- and L-states cause a de-shielding of the 15N nitrogen compared to the dark state. This indicates a stepwise stronger interaction with the counterion as the Schiff base proton moves away from the Schiff base and comes closer to the D116 in the transition from K- to L-state and approaches the proton transfer step during the M-state formation. In the O-state, the retinal was found to be in the all-trans conformation but differed to the dark state in the C13, C20, and Schiff base nitrogen chemical shifts. The largest effect (9 ppm) was observed for the Schiff base nitrogen, which could be explained by the effect of the positive charge of bound Na+ near the Schiff base in the O-state, coordinated by N112 and D116 as observed in the O-state crystal structure in the pentameric form. The structural change at the opsin followed the retinal isomerization and the energy transfer from the chromophore to the surrounding were also investigated in this thesis using various amino acids labeling schemes. Moreover, 1H-13C hNOE in combination with CE-DNP was applied to probe the dynamics of retinylidene methyl groups and 23Na MAS NMR was employed to detect the bound sodium ion at the protomer interface in KR2 dark state.

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A single mutation converts bacterial Na⁺‐transporting rhodopsin into an H⁺ transporter

Cited by 16 publications

References 30 publications

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport

Sodium Energetic Cycle in the Natronophilic Bacterium Thioalkalivibrio versutus

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

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A single mutation converts bacterial Na+‐transporting rhodopsin into an H+ transporter

Cited by 16 publications

References 30 publications

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport

Sodium Energetic Cycle in the Natronophilic Bacterium Thioalkalivibrio versutus

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

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A single mutation converts bacterial Na⁺‐transporting rhodopsin into an H⁺ transporter