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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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Tentative scheme for internal ion transferand binding that accountsfor the observed uptake of H+ from the bulk and its release.The top surface of the schematic representation of the protein isthe cytoplasmic side. For the sake of clarity, not all detected intermediatesare shown. The top sequence after the branch is the “Na+-independent” cycle and the bottom the “Na+-dependent” cycle. SBH+ and SB refer tothe protonated and unprotonated retinal Schiff base, respectively.“Asp” refers to the proton acceptor group and the ionizablepart of the Na+ binding site, without commitment as towhether it is Asp116, Asp251, or both. The dashed circle is a postulatedproton binding site analogous to the proton release site of bacteriorhodopsin,made necessary by the observation of proton release in the O stateof the Na+-dependent cycle.
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fig10: Tentative scheme for internal ion transferand binding that accountsfor the observed uptake of H+ from the bulk and its release.The top surface of the schematic representation of the protein isthe cytoplasmic side. For the sake of clarity, not all detected intermediatesare shown. The top sequence after the branch is the “Na+-independent” cycle and the bottom the “Na+-dependent” cycle. SBH+ and SB refer tothe protonated and unprotonated retinal Schiff base, respectively.“Asp” refers to the proton acceptor group and the ionizablepart of the Na+ binding site, without commitment as towhether it is Asp116, Asp251, or both. The dashed circle is a postulatedproton binding site analogous to the proton release site of bacteriorhodopsin,made necessary by the observation of proton release in the O stateof the Na+-dependent cycle.

Mentions: Taking advantage of the considerable body of mechanistic informationfrom bacteriorhodopsin and similar proteins, we propose a minimalscheme that is consistent with all the data (Figure 10). We need to assume three sites: the retinal Schiff base,a counterion that is also part of a binding site for H+ and Na+, and an additional site for H+ thatcould be analogous to the extracellular proton release site of BR.49,68,75,76 A model with fewer sites cannot be reconciled with the observations.The first steps in the photocycle do not depend on Na+.For the K state of KR2, this was explicitly shown.77 M formation is very rapid, on a time scale of a few microseconds,suggesting the existence of a ready acceptor for the Schiff base proton.Asp116 and Asp251 are the likely candidates, but a state in whichthis could be examined using FTIR spectroscopy could not be trapped.The initial H+ release occurs at approximately the sametime in the presence and absence of Na+ (Figure 5C). It is delayed compared to M rise (Figure 5A), suggesting that the proton is not directly fromthe Schiff base, providing further support for the involvement ofa proton acceptor. This means also that there are two M states: onebefore proton release and another after, as in BR.49,68,75


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)

Tentative scheme for internal ion transferand binding that accountsfor the observed uptake of H+ from the bulk and its release.The top surface of the schematic representation of the protein isthe cytoplasmic side. For the sake of clarity, not all detected intermediatesare shown. The top sequence after the branch is the “Na+-independent” cycle and the bottom the “Na+-dependent” cycle. SBH+ and SB refer tothe protonated and unprotonated retinal Schiff base, respectively.“Asp” refers to the proton acceptor group and the ionizablepart of the Na+ binding site, without commitment as towhether it is Asp116, Asp251, or both. The dashed circle is a postulatedproton binding site analogous to the proton release site of bacteriorhodopsin,made necessary by the observation of proton release in the O stateof the Na+-dependent cycle.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Tentative scheme for internal ion transferand binding that accountsfor the observed uptake of H+ from the bulk and its release.The top surface of the schematic representation of the protein isthe cytoplasmic side. For the sake of clarity, not all detected intermediatesare shown. The top sequence after the branch is the “Na+-independent” cycle and the bottom the “Na+-dependent” cycle. SBH+ and SB refer tothe protonated and unprotonated retinal Schiff base, respectively.“Asp” refers to the proton acceptor group and the ionizablepart of the Na+ binding site, without commitment as towhether it is Asp116, Asp251, or both. The dashed circle is a postulatedproton binding site analogous to the proton release site of bacteriorhodopsin,made necessary by the observation of proton release in the O stateof the Na+-dependent cycle.
Mentions: Taking advantage of the considerable body of mechanistic informationfrom bacteriorhodopsin and similar proteins, we propose a minimalscheme that is consistent with all the data (Figure 10). We need to assume three sites: the retinal Schiff base,a counterion that is also part of a binding site for H+ and Na+, and an additional site for H+ thatcould be analogous to the extracellular proton release site of BR.49,68,75,76 A model with fewer sites cannot be reconciled with the observations.The first steps in the photocycle do not depend on Na+.For the K state of KR2, this was explicitly shown.77 M formation is very rapid, on a time scale of a few microseconds,suggesting the existence of a ready acceptor for the Schiff base proton.Asp116 and Asp251 are the likely candidates, but a state in whichthis could be examined using FTIR spectroscopy could not be trapped.The initial H+ release occurs at approximately the sametime in the presence and absence of Na+ (Figure 5C). It is delayed compared to M rise (Figure 5A), suggesting that the proton is not directly fromthe Schiff base, providing further support for the involvement ofa proton acceptor. This means also that there are two M states: onebefore proton release and another after, as in BR.49,68,75

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