Cl− Concentration Dependence of Photovoltage Generation by Halorhodopsin from Halobacterium salinarum

Muneyuki, Eiro; Shibazaki, C.; Wada, Yoichiro; Yakushizin, Manabu; Ohtani, Hiroyuki

doi:10.1016/s0006-3495(02)73941-6

Cited by 8 publications

(9 citation statements)

References 29 publications

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“…3) The lifetime of the 13-cis conformation has been determined while blocking anion transport by high chloride concentrations (20). In 5 M Cl Ϫ solvent conditions, hR no longer transfers anions after photoexcitation, but relaxes from the 13-cis back to the all-trans form in about 100 ms (dotted arrow in Fig.…”

Section: Discussionmentioning

confidence: 99%

The Mechanism of Photo-energy Storage in the Halorhodopsin Chloride Pump

Pfisterer

Gruia

Fischer

2009

Journal of Biological Chemistry

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The light-driven pump Halorhodopsin (hR) uses the energy stored in an initial meta-stable state (K), in which the bound retinal chromophore has been photoisomerized from all-trans to 13-cis, to drive the translocation of one chloride anion across the membrane. Thus far it was unclear whether retinal twisting or charge separation between the positive Schiff base of the retinal and the chloride anion is the primary mechanism of energy storage. Here, combined quantum mechanical/molecular mechanical (QM/MM) simulations show that the energy is predominantly stored by charge separation. However, a large variability in retinal twisting is observed, thus reconciling the contradictory hypotheses for storage. Surprisingly, the energy stored in the K-state amounts to only one-fifth of the photon energy. We explain why the protein cannot store more: even though this would accelerate chloride pumping, raising the K-state also reduces the relative energy barriers against unproductive decay, in particular via the premature cis to trans backisomerization. Indeed, the protein has maximized its storage so that the back-isomerization barriers are just high enough (>18 kcal/mol) to keep the decay rate (1/100 ms) slower than the remaining photocycle (1/20 ms). This need to stabilize the captured photon-energy until it can be used in subsequent steps is inherent to light-driven proteins.Light driven pumps such as halorhodopsin (hR) 2 stand apart from other energy-driven transporters. To them, the primary source of energy is not permanently available, unlike energy stored in the form of chemical bonds (for example, ATP in the case of the Na ϩ /K ϩ -ATPase (1)) or in electrochemical transmembrane gradients (like the Na ϩ /glucose symport transporter (2)). Thus, they must be able to first harvest the energy of the photon, before converting that energy into work. The harvesting of the fleeting photon necessitates converting the photon energy into a meta-stable form of molecular energy, and to prevent the dissipation of this molecular energy until it can be subsequently used to power the pumping mechanism. In other words, the protein must be able to simultaneously store and stabilize the energy. For the absorption of light, proteins employ a chromophoric ligand or group, which changes from one state to another upon photon absorption. This transition is unfavorable and slow in the electronic ground-state, but is favored when the chromophore has been excited by the photon. After the return of the system to the electronic ground-state (within picoseconds in the case of hR (3)), the chromophore relaxes either to the starting ground-state conformation (thus reducing the quantum yield) or to a meta-stable high-energy state (with a quantum yield of 34% in the case of hR (3)). The energy stored in this meta-stable form relative to the groundstate form is what powers the subsequent pumping steps (Fig. 1A). Understanding how this energy is stored thus constitutes an essential aspect of the mechanism and has been the focus of studies on visual rh...

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Section: Discussionmentioning

confidence: 99%

The Mechanism of Photo-energy Storage in the Halorhodopsin Chloride Pump

Pfisterer

Gruia

Fischer

2009

Journal of Biological Chemistry

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“…Simultaneous absorption kinetic and electric signal measurements, on oriented gel samples, allowed the calculation of the time courses of the photocycle intermediates and, at the same time, of their electrogenicities (Ludmann et al, 2000). Another attempt to interpret the relation of the electric signals to the photocycle was made by measuring the photocycle in suspension and the electric signals by adsorbing HR containing membranes onto a thin polymer film (Okuno et al, 1999;Muneyuki et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

The Nitrate Transporting Photochemical Reaction Cycle of the Pharaonis Halorhodopsin

Bálint

Lakatos

Ganea

et al. 2004

Biophysical Journal

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Time-resolved spectroscopy, absorption kinetic and electric signal measurement techniques were used to study the nitrate transporting photocycle of the pharaonis halorhodopsin. The spectral titration reveals two nitrate-binding constants, assigned to two independent binding sites. The high-affinity binding site (K(a) = 11 mM) contributes to the appearance of the nitrate transporting photocycle, whereas the low-affinity constant (having a K(a) of approximately 7 M) slows the last decay process in the photocycle. Although the spectra of the intermediates are not the same as those found in the chloride transporting photocycle, the sequence of the intermediates and the energy diagrams are similar. The differences in spectra and energy levels can be attributed to the difference in the size of the transported chloride or nitrate. Electric signal measurements show that a charge is transferred across the membrane during the photocycle, as expected. A new observation is an apparent release and rebinding of a small fraction of the retinal, inside the retinal pocket, during the photocycle. The release occurs during the N-to-O transition, whereas the rebinding happens in several seconds, well after the other steps of the photocycle are over.

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“…29 It is reasonable to consider that the rate of the anion uptake process is proportional to ion occupancy at this binding site (site v118). 31 In this case, the rate of anion uptake, k, can be given by the equation k = γ/(1 + K ext /c), where c is the bromide ion concentration, K ext is the dissociation constant of the bromide ion at site v118, and γ is the rate constant that would be observed at the full bromide ion occupancy at site v118. The two dashed lines in Figure 6a represent the decay time constants that were evaluated using different K ext values (0.59 and 3.0 M) but the same γ value (0.25 ms −1 ).…”

Section: ■ Resultsmentioning

confidence: 99%

“…A previous crystallographic study of p HR in the presence of bromide ions has suggested that the structure of the trimeric assembly of p HR is not described with perfect 3-fold rotational symmetry; in particular, the occupancy of the halide ion at a binding site near the main chain of Val118 (site v118), which exists in the anion uptake pathway from the extracellular side, is considerably lower in one subunit (C subunit) as compared with those in the other subunits . It is reasonable to consider that the rate of the anion uptake process is proportional to ion occupancy at this binding site (site v118) . In this case, the rate of anion uptake, k , can be given by the equation k = γ/(1 + K ext / c ), where c is the bromide ion concentration, K ext is the dissociation constant of the bromide ion at site v118, and γ is the rate constant that would be observed at the full bromide ion occupancy at site v118.…”

Section: Resultsmentioning

confidence: 99%

Three-Step Isomerization of the Retinal Chromophore during the Anion Pumping Cycle of Halorhodopsin

et al. 2018

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The anion pumping cycle of halorhodopsin from Natronomonas pharaonis ( pHR) is initiated when the all- trans/15- anti isomer of retinal is photoisomerized into the 13- cis/15- anti configuration. A recent crystallographic study suggested that a reaction state with 13- cis/15- syn retinal occurred during the anion release process, i.e., after the N state with the 13- cis/15- anti retinal and before the O state with all- trans/15- anti retinal. In this study, we investigated the retinal isomeric composition in a long-living reaction state at various bromide ion concentrations. It was found that the 13- cis isomer (HR'), in which the absorption spectrum was blue-shifted by ∼8 nm compared with that of the trans isomer (HR), accumulated significantly when a cold suspension of pHR-rich claret membranes in 4 M NaBr was illuminated with continuous light. Analysis of flash-induced absorption changes suggested that the branching of the trans photocycle into the 13- cis isomer (HR') occurs during the decay of an O-like state (O') with 13- cis/15- syn retinal; i.e., O' can decay to either HR' or O with all- trans/15- anti retinal. The efficiency of the branching reaction was found to be dependent on the bromide ion concentration. At a very high bromide ion concentration, the anion pumping cycle is described by the schemeHR -( hν) → K → L ↔ L ↔ N ↔ N' ↔ O' ↔ HR' ↔HR. At a low bromide ion concentration, on the other hand, O' decays into HR via O.

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Cl− Concentration Dependence of Photovoltage Generation by Halorhodopsin from Halobacterium salinarum

Cited by 8 publications

References 29 publications

The Mechanism of Photo-energy Storage in the Halorhodopsin Chloride Pump

The Mechanism of Photo-energy Storage in the Halorhodopsin Chloride Pump

The Nitrate Transporting Photochemical Reaction Cycle of the Pharaonis Halorhodopsin

Three-Step Isomerization of the Retinal Chromophore during the Anion Pumping Cycle of Halorhodopsin

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