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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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Light-induced pH changes produced by GLR assayedwith pyranine(pH 7.2–7.4). (A) In 100 mM KCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 590 nm. Protonrelease occurs with two time constants, 0.7 and 9.9 ms; the subsequentproton uptake with one (430 ms) and slow release one (2.6 ± 0.1s). The decay of M (ΔA at 410 nm) and the riseof the red-shifted intermediate (ΔA at 590nm) occurred with a time constant of 400 ms, similar to that of H+ uptake. The decay of ΔA at 590 nmoccurs with a time constant of 2.7 s, similar to that of slow protonrelease. (B) In 100 mM NaCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 450 nm; 4,ΔA at 590 nm. Proton release occurs with atime constant of ∼1 ms and uptake with a time constant of 50ms. (C) Comparison of the pyranine response in 100 mM KCl and 100mM NaCl. A decrease in pyranine absorbance corresponds to proton release.
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fig5: Light-induced pH changes produced by GLR assayedwith pyranine(pH 7.2–7.4). (A) In 100 mM KCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 590 nm. Protonrelease occurs with two time constants, 0.7 and 9.9 ms; the subsequentproton uptake with one (430 ms) and slow release one (2.6 ± 0.1s). The decay of M (ΔA at 410 nm) and the riseof the red-shifted intermediate (ΔA at 590nm) occurred with a time constant of 400 ms, similar to that of H+ uptake. The decay of ΔA at 590 nmoccurs with a time constant of 2.7 s, similar to that of slow protonrelease. (B) In 100 mM NaCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 450 nm; 4,ΔA at 590 nm. Proton release occurs with atime constant of ∼1 ms and uptake with a time constant of 50ms. (C) Comparison of the pyranine response in 100 mM KCl and 100mM NaCl. A decrease in pyranine absorbance corresponds to proton release.

Mentions: Wemeasured the kinetics of transient H+ concentration changesin the bulk after flash photoexcitation, using pyranine as a pH sensitiveprobe.34,49 The increase in absorbance at 455 nm fromthe dye (after subtraction of the trace from a sample without thedye) corresponds to proton uptake, whereas a decrease indicates protonrelease. As shown in Figure 5A, in the absenceof Na+, formation of the intermediate M, with two timeconstants of 35 μs (60%) and 150 μs (40%), is followedby proton release with a time constant of ∼2 ms. The decayof M and the formation of N/O-like states, with τ ≈ 400ms, virtually coincide with the uptake of protons (that reverses theearlier release and causes net alkalinization of the bulk). At theend of the cycle, the decay of the long-lived O state with τ= 2.7 s is accompanied by a coincident release (with τ = 2.6± 0.1 s) of the net proton gained earlier during the decay ofM and formation of N/O.


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)

Light-induced pH changes produced by GLR assayedwith pyranine(pH 7.2–7.4). (A) In 100 mM KCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 590 nm. Protonrelease occurs with two time constants, 0.7 and 9.9 ms; the subsequentproton uptake with one (430 ms) and slow release one (2.6 ± 0.1s). The decay of M (ΔA at 410 nm) and the riseof the red-shifted intermediate (ΔA at 590nm) occurred with a time constant of 400 ms, similar to that of H+ uptake. The decay of ΔA at 590 nmoccurs with a time constant of 2.7 s, similar to that of slow protonrelease. (B) In 100 mM NaCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 450 nm; 4,ΔA at 590 nm. Proton release occurs with atime constant of ∼1 ms and uptake with a time constant of 50ms. (C) Comparison of the pyranine response in 100 mM KCl and 100mM NaCl. A decrease in pyranine absorbance corresponds to proton release.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Light-induced pH changes produced by GLR assayedwith pyranine(pH 7.2–7.4). (A) In 100 mM KCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 590 nm. Protonrelease occurs with two time constants, 0.7 and 9.9 ms; the subsequentproton uptake with one (430 ms) and slow release one (2.6 ± 0.1s). The decay of M (ΔA at 410 nm) and the riseof the red-shifted intermediate (ΔA at 590nm) occurred with a time constant of 400 ms, similar to that of H+ uptake. The decay of ΔA at 590 nmoccurs with a time constant of 2.7 s, similar to that of slow protonrelease. (B) In 100 mM NaCl: 1, pyranine response; 2, ΔA at 410 nm; 3, ΔA at 450 nm; 4,ΔA at 590 nm. Proton release occurs with atime constant of ∼1 ms and uptake with a time constant of 50ms. (C) Comparison of the pyranine response in 100 mM KCl and 100mM NaCl. A decrease in pyranine absorbance corresponds to proton release.
Mentions: Wemeasured the kinetics of transient H+ concentration changesin the bulk after flash photoexcitation, using pyranine as a pH sensitiveprobe.34,49 The increase in absorbance at 455 nm fromthe dye (after subtraction of the trace from a sample without thedye) corresponds to proton uptake, whereas a decrease indicates protonrelease. As shown in Figure 5A, in the absenceof Na+, formation of the intermediate M, with two timeconstants of 35 μs (60%) and 150 μs (40%), is followedby proton release with a time constant of ∼2 ms. The decayof M and the formation of N/O-like states, with τ ≈ 400ms, virtually coincide with the uptake of protons (that reverses theearlier release and causes net alkalinization of the bulk). At theend of the cycle, the decay of the long-lived O state with τ= 2.7 s is accompanied by a coincident release (with τ = 2.6± 0.1 s) of the net proton gained earlier during the decay ofM and formation of N/O.

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