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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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Kinetics of the GLR photocycle in 100 mM NaCl in D2O(pD 7.6). (A) Absorption changes at four selected wavelengths. (B)Initial, difference spectrum “K minus initial GLR” (1μs after the flash). The numerals 1 and 2 denote the first twocomponents of the decay of K to an M-like and X470 intermediateswith τ1 = 7.7 μs and τ2 =48 μs. (C and D) Difference spectra of the subsequent third,fourth, fifth, sixth, and seventh transitions that occur with timesof 124 μs, 0.9 ms, 4.3 ms, 12 ms, and 54 ms, respectively.
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fig4: Kinetics of the GLR photocycle in 100 mM NaCl in D2O(pD 7.6). (A) Absorption changes at four selected wavelengths. (B)Initial, difference spectrum “K minus initial GLR” (1μs after the flash). The numerals 1 and 2 denote the first twocomponents of the decay of K to an M-like and X470 intermediateswith τ1 = 7.7 μs and τ2 =48 μs. (C and D) Difference spectra of the subsequent third,fourth, fifth, sixth, and seventh transitions that occur with timesof 124 μs, 0.9 ms, 4.3 ms, 12 ms, and 54 ms, respectively.

Mentions: Therecovery of the initial state in 100 mM NaCl is ∼50-foldfaster than in the Na+-independent cycle (∼50 msvs 2.6 s). To better resolve the rapid early steps involving the formationof M and X470, we used D2O instead of H2O, which slows them by a factor of ∼2.5. Subsequentstages are less affected. In 100 mM NaCl, the kinetics of M decayin D2O slows by <1.4-fold, much lower than the valueof 8-fold in the absence of Na+, suggesting that bindingof a sodium ion facilitates reprotonation of the Schiff base in theM to N transition and determines its rate. Figure 4A shows the kinetics at selected wavelengths. In panels B–D,the spectra of the initial (first) absorption changes produced bylight (labeled Initial) and the seven subsequent kinetic steps obtainedfrom a global fit are shown. The spectra, given here as differencesbetween consecutive intermediates, represent conversion of each intermediatestate into the next. In the first transition (τ1 =7.7 μs), a fraction (∼30%) of K with a maximum at 550nm is replaced by the short wavelength M-like intermediate, with amaximum at ≤400 nm. In the subsequent step (τ2 = 48 μs), most of the remaining K is replaced by X470. In the third step (τ3 = 124 μs), the M-likestate and the rest of K disappear and a red-shifted species (maximumin the difference spectrum is at 550 nm) is produced. The fourth andfifth steps involve transitions of the X470 intermediatewith τ4 = 0.9 ms and τ5 = 4.3 msinto species designated as N and O, with maxima in the differenceabsorption spectra at 570 and 600 nm, respectively. The initial stateis recovered in the sixth and seventh kinetic steps with τ6 = 12 ms and τ7 = 54 ms through decay ofO. The Na+-dependent and Na+-independent cyclesdiffer therefore mainly in the time constants of the interconversions.The mixtures of species associated with single kinetic steps arisemost likely from back reactions that produce transient equilibriaof several states, as in other retinal proteins.44−48


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)

Kinetics of the GLR photocycle in 100 mM NaCl in D2O(pD 7.6). (A) Absorption changes at four selected wavelengths. (B)Initial, difference spectrum “K minus initial GLR” (1μs after the flash). The numerals 1 and 2 denote the first twocomponents of the decay of K to an M-like and X470 intermediateswith τ1 = 7.7 μs and τ2 =48 μs. (C and D) Difference spectra of the subsequent third,fourth, fifth, sixth, and seventh transitions that occur with timesof 124 μs, 0.9 ms, 4.3 ms, 12 ms, and 54 ms, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Kinetics of the GLR photocycle in 100 mM NaCl in D2O(pD 7.6). (A) Absorption changes at four selected wavelengths. (B)Initial, difference spectrum “K minus initial GLR” (1μs after the flash). The numerals 1 and 2 denote the first twocomponents of the decay of K to an M-like and X470 intermediateswith τ1 = 7.7 μs and τ2 =48 μs. (C and D) Difference spectra of the subsequent third,fourth, fifth, sixth, and seventh transitions that occur with timesof 124 μs, 0.9 ms, 4.3 ms, 12 ms, and 54 ms, respectively.
Mentions: Therecovery of the initial state in 100 mM NaCl is ∼50-foldfaster than in the Na+-independent cycle (∼50 msvs 2.6 s). To better resolve the rapid early steps involving the formationof M and X470, we used D2O instead of H2O, which slows them by a factor of ∼2.5. Subsequentstages are less affected. In 100 mM NaCl, the kinetics of M decayin D2O slows by <1.4-fold, much lower than the valueof 8-fold in the absence of Na+, suggesting that bindingof a sodium ion facilitates reprotonation of the Schiff base in theM to N transition and determines its rate. Figure 4A shows the kinetics at selected wavelengths. In panels B–D,the spectra of the initial (first) absorption changes produced bylight (labeled Initial) and the seven subsequent kinetic steps obtainedfrom a global fit are shown. The spectra, given here as differencesbetween consecutive intermediates, represent conversion of each intermediatestate into the next. In the first transition (τ1 =7.7 μs), a fraction (∼30%) of K with a maximum at 550nm is replaced by the short wavelength M-like intermediate, with amaximum at ≤400 nm. In the subsequent step (τ2 = 48 μs), most of the remaining K is replaced by X470. In the third step (τ3 = 124 μs), the M-likestate and the rest of K disappear and a red-shifted species (maximumin the difference spectrum is at 550 nm) is produced. The fourth andfifth steps involve transitions of the X470 intermediatewith τ4 = 0.9 ms and τ5 = 4.3 msinto species designated as N and O, with maxima in the differenceabsorption spectra at 570 and 600 nm, respectively. The initial stateis recovered in the sixth and seventh kinetic steps with τ6 = 12 ms and τ7 = 54 ms through decay ofO. The Na+-dependent and Na+-independent cyclesdiffer therefore mainly in the time constants of the interconversions.The mixtures of species associated with single kinetic steps arisemost likely from back reactions that produce transient equilibriaof several states, as in other retinal proteins.44−48

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