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Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

Balashov SP, Imasheva ES, Dioumaev AK, Wang JM, Jung KH, Lanyi JK - Biochemistry (2014)

Bottom Line: However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+).A greater concentration of Na(+) is needed for switching the reaction path at lower pH.Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.

ABSTRACT
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

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Effect of sodiumchloride on the kinetics of the GLR photocycleat two selected wavelengths, 590 nm, representing formation and decayof the red-shifted intermediate(s) K and O, and 410 nm, tracking theblue-shifted intermediate(s) M. (A) Traces 1–8 are the absorptionchanges at 0.3 μM, 100 μM, 300 μM, 1 mM, 3 mM, 10mM, 30 mM, and 100 mM NaCl. (B) Rate constant of the decay of theblue-shifted intermediate vs NaCl concentration (in a single-componentfit; k0 is reciprocal of the half-decaytime at 410 nm at 0.3 μM NaCl). (C) Decrease in the amplitudeof absorption changes at 590 nm from the long-lived O-like intermediate(time constant of 2.6 s) upon addition of NaCl, which indicates aswitch from a “Na+-independent” to a “Na+-dependent” photocycle. The data were fit with theequation ΔA590([Na+])= ΔA0/(1 + K[Na+]) (see the text), from which it was determined that the Na+ concentration at which 50% of the molecules proceed throughthe sodium ion-dependent cycle is K–1 = 60 ± 7 μM.
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fig3: Effect of sodiumchloride on the kinetics of the GLR photocycleat two selected wavelengths, 590 nm, representing formation and decayof the red-shifted intermediate(s) K and O, and 410 nm, tracking theblue-shifted intermediate(s) M. (A) Traces 1–8 are the absorptionchanges at 0.3 μM, 100 μM, 300 μM, 1 mM, 3 mM, 10mM, 30 mM, and 100 mM NaCl. (B) Rate constant of the decay of theblue-shifted intermediate vs NaCl concentration (in a single-componentfit; k0 is reciprocal of the half-decaytime at 410 nm at 0.3 μM NaCl). (C) Decrease in the amplitudeof absorption changes at 590 nm from the long-lived O-like intermediate(time constant of 2.6 s) upon addition of NaCl, which indicates aswitch from a “Na+-independent” to a “Na+-dependent” photocycle. The data were fit with theequation ΔA590([Na+])= ΔA0/(1 + K[Na+]) (see the text), from which it was determined that the Na+ concentration at which 50% of the molecules proceed throughthe sodium ion-dependent cycle is K–1 = 60 ± 7 μM.

Mentions: Therise or decay of the photocycle intermediates, if they are dependenton the bulk Na+ concentration, can serve as intrinsic reportersof transient Na+ binding. We gained insight into the dynamicsof sodium ion transport and the possible location of the principalbinding site by exploring the Na+ concentration dependenceof the photocycle transitions monitored at characteristic wavelengths(Figure 3A). It appears that addition of 0.1,1, and 3 mM NaCl progressively replaces the slowly forming and slowlydecaying red-shifted (O-like) intermediate with a rapidly rising anddecaying O-like intermediate (Figure 3A, traces2–4, respectively) and shortens the lifetime of the M stateby linearly increasing the rate constant of its decay (Figure 3B). The existence of two parallel pathways is evidentfrom traces 2 and 3 of Figure 3A, which showthat the rapid and slow O intermediates coexist. We refer to the twocycles as “Na+-dependent” and “Na+-independent”, respectively. The transition from theNa+-independent photocycle to the Na+-dependentphotocycle is best characterized by the pH dependence of the yieldof the long-lived O-like intermediate assayed as absorption changesat 590 nm (Figure 3C). The data can be fitby a simple equation derived from a scheme for the photocycle thatbranches at M, where some of the molecules in the blue-shifted intermediatesM and X470 undergo transitions to the red-shifted intermediates.The time constants are k0 and kb[Na+] for the Na+-independentand Na+-dependent branches, respectively, where kb is the rate constant for Na+ bindingin M. This leads to the following equation: ΔA590 = ΔA0/(1 + K[Na+]), where ΔA590 is the maximal amplitude of the absorption change at 590nm for the long-lived O intermediate, K = kb/k0, and ΔA0 is the ΔA590 in the absence of Na+. The concentration of Na+ at which the level of accumulation of the long-lived O intermediateis decreased by half is ∼60 μM (Figure 3C). We note that the value of 60 μM is not the equilibriumbinding constant for Na+, but the Na+ concentrationat which the photocycle branching is at its midpoint. Its relativelylow value indicates a high affinity of the transiently formed bindingsite for sodium. Further additions of NaCl not only completely eliminatethe long-lived O intermediate but also continue to accelerate thedecay of the blue-shifted intermediate M and the corresponding formationof the red-shifted state(s), N/O (Figure 3A,traces 5–8). The rate of M decay is accelerated by 3 ordersof magnitude in 100 mM NaCl and continues to accelerate up to 1 MNa+ (latter not shown). The linear dependence of the rateof its conversion on Na+ concentration (Figure 3B) indicates that entry of Na+ into atransiently formed binding site, which arises during the photocycle,becomes rate-limiting. The subsequent reactions of the photocycleare similarly affected (Figure 3A).


Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

Balashov SP, Imasheva ES, Dioumaev AK, Wang JM, Jung KH, Lanyi JK - Biochemistry (2014)

Effect of sodiumchloride on the kinetics of the GLR photocycleat two selected wavelengths, 590 nm, representing formation and decayof the red-shifted intermediate(s) K and O, and 410 nm, tracking theblue-shifted intermediate(s) M. (A) Traces 1–8 are the absorptionchanges at 0.3 μM, 100 μM, 300 μM, 1 mM, 3 mM, 10mM, 30 mM, and 100 mM NaCl. (B) Rate constant of the decay of theblue-shifted intermediate vs NaCl concentration (in a single-componentfit; k0 is reciprocal of the half-decaytime at 410 nm at 0.3 μM NaCl). (C) Decrease in the amplitudeof absorption changes at 590 nm from the long-lived O-like intermediate(time constant of 2.6 s) upon addition of NaCl, which indicates aswitch from a “Na+-independent” to a “Na+-dependent” photocycle. The data were fit with theequation ΔA590([Na+])= ΔA0/(1 + K[Na+]) (see the text), from which it was determined that the Na+ concentration at which 50% of the molecules proceed throughthe sodium ion-dependent cycle is K–1 = 60 ± 7 μM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263435&req=5

fig3: Effect of sodiumchloride on the kinetics of the GLR photocycleat two selected wavelengths, 590 nm, representing formation and decayof the red-shifted intermediate(s) K and O, and 410 nm, tracking theblue-shifted intermediate(s) M. (A) Traces 1–8 are the absorptionchanges at 0.3 μM, 100 μM, 300 μM, 1 mM, 3 mM, 10mM, 30 mM, and 100 mM NaCl. (B) Rate constant of the decay of theblue-shifted intermediate vs NaCl concentration (in a single-componentfit; k0 is reciprocal of the half-decaytime at 410 nm at 0.3 μM NaCl). (C) Decrease in the amplitudeof absorption changes at 590 nm from the long-lived O-like intermediate(time constant of 2.6 s) upon addition of NaCl, which indicates aswitch from a “Na+-independent” to a “Na+-dependent” photocycle. The data were fit with theequation ΔA590([Na+])= ΔA0/(1 + K[Na+]) (see the text), from which it was determined that the Na+ concentration at which 50% of the molecules proceed throughthe sodium ion-dependent cycle is K–1 = 60 ± 7 μM.
Mentions: Therise or decay of the photocycle intermediates, if they are dependenton the bulk Na+ concentration, can serve as intrinsic reportersof transient Na+ binding. We gained insight into the dynamicsof sodium ion transport and the possible location of the principalbinding site by exploring the Na+ concentration dependenceof the photocycle transitions monitored at characteristic wavelengths(Figure 3A). It appears that addition of 0.1,1, and 3 mM NaCl progressively replaces the slowly forming and slowlydecaying red-shifted (O-like) intermediate with a rapidly rising anddecaying O-like intermediate (Figure 3A, traces2–4, respectively) and shortens the lifetime of the M stateby linearly increasing the rate constant of its decay (Figure 3B). The existence of two parallel pathways is evidentfrom traces 2 and 3 of Figure 3A, which showthat the rapid and slow O intermediates coexist. We refer to the twocycles as “Na+-dependent” and “Na+-independent”, respectively. The transition from theNa+-independent photocycle to the Na+-dependentphotocycle is best characterized by the pH dependence of the yieldof the long-lived O-like intermediate assayed as absorption changesat 590 nm (Figure 3C). The data can be fitby a simple equation derived from a scheme for the photocycle thatbranches at M, where some of the molecules in the blue-shifted intermediatesM and X470 undergo transitions to the red-shifted intermediates.The time constants are k0 and kb[Na+] for the Na+-independentand Na+-dependent branches, respectively, where kb is the rate constant for Na+ bindingin M. This leads to the following equation: ΔA590 = ΔA0/(1 + K[Na+]), where ΔA590 is the maximal amplitude of the absorption change at 590nm for the long-lived O intermediate, K = kb/k0, and ΔA0 is the ΔA590 in the absence of Na+. The concentration of Na+ at which the level of accumulation of the long-lived O intermediateis decreased by half is ∼60 μM (Figure 3C). We note that the value of 60 μM is not the equilibriumbinding constant for Na+, but the Na+ concentrationat which the photocycle branching is at its midpoint. Its relativelylow value indicates a high affinity of the transiently formed bindingsite for sodium. Further additions of NaCl not only completely eliminatethe long-lived O intermediate but also continue to accelerate thedecay of the blue-shifted intermediate M and the corresponding formationof the red-shifted state(s), N/O (Figure 3A,traces 5–8). The rate of M decay is accelerated by 3 ordersof magnitude in 100 mM NaCl and continues to accelerate up to 1 MNa+ (latter not shown). The linear dependence of the rateof its conversion on Na+ concentration (Figure 3B) indicates that entry of Na+ into atransiently formed binding site, which arises during the photocycle,becomes rate-limiting. The subsequent reactions of the photocycleare similarly affected (Figure 3A).

Bottom Line: However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+).A greater concentration of Na(+) is needed for switching the reaction path at lower pH.Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.

ABSTRACT
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

Show MeSH
Related in: MedlinePlus