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

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.

Figure 1

Light-induced pH changes in a suspension of E. coli cells with G. limnaea rhodopsin expressed and reconstituted with all-trans-retinal: trace 1, absence of pH change in sodium free medium [in 100 mM KCl (pH 7.5)]; trace 2, proton uptake (alkalinization) in 100 mM NaCl; trace 3, same as trace 2 but after addition of a protonophore (50 μM CCCP), which increases the rate and extent of passive proton influx.

Figure 2

Flash-induced absorption changes of GLR at pH 8.5 in 10 mM KCl at several characteristic wavelengths, 410, 510, 550, and 590 nm. The concentration of NaCl is <1 μM. The traces were globally fit with six kinetic components with time constants shown at the top.

Figure 3

Effect of sodium chloride on the kinetics of the GLR photocycle at two selected wavelengths, 590 nm, representing formation and decay of the red-shifted intermediate(s) K and O, and 410 nm, tracking the blue-shifted intermediate(s) M. (A) Traces 1–8 are the absorption changes at 0.3 μM, 100 μM, 300 μM, 1 mM, 3 mM, 10 mM, 30 mM, and 100 mM NaCl. (B) Rate constant of the decay of the blue-shifted intermediate vs NaCl concentration (in a single-component fit; k₀ is reciprocal of the half-decay time at 410 nm at 0.3 μM NaCl). (C) Decrease in the amplitude of absorption changes at 590 nm from the long-lived O-like intermediate (time constant of 2.6 s) upon addition of NaCl, which indicates a switch from a “Na⁺-independent” to a “Na⁺-dependent” photocycle. The data were fit with the equation 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 through the sodium ion-dependent cycle is K^–1 = 60 ± 7 μM.

Figure 4

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” (1 μs after the flash). The numerals 1 and 2 denote the first two components of the decay of K to an M-like and X₄₇₀ intermediates with 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 times of 124 μs, 0.9 ms, 4.3 ms, 12 ms, and 54 ms, respectively.

Figure 5

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

Figure 6

Light minus dark FTIR spectra of photostationary states at 270 K: spectrum 1, in the absence of NaCl (<1 μM); spectrum 2, in 150 mM NaCl (pH8.6). The ethylenic stretch, the fingerprint, and the HOOP regions of spectra in the infrared and the corresponding spectra in the visible range of the same samples (not shown) indicate that the intermediate trapped is like the O state of bacteriorhodopsin. The Na⁺ dependence of the C=O stretch region is discussed in the text.

Figure 7

pH dependence of transient absorption changes of GLR in the presence of 100 mM NaCl at selected wavelengths: (A) 410 nm and (B) 590 and 510 nm. Numerals 1–4 correspond to pH 8.3, 6.1, 5.1, and 3.5, respectively.

Figure 8

pH dependence of the absorption spectra of GLR and its D116N and D251N mutants. (A) Chromophore absorption bands of (1) the wild type (WT) at pH 8.0, (2) WT at pH 3.3, (3) D116N at pH 7.l, and (4) D251N at pH 8.0. (B) Titration of WT from pH 9.0 to 3.0 in 100 mM KCl (⊞) and 100 mM NaCl (●). (C) Difference spectrum from a decrease in pH from 7.0 to 3.5 in (1) WT, (2) D251N, and (3) D116N and (4 and 5) absorption changes that accompany recovery of the initial state in the photocycle in the absence of Na⁺ (4, ●) and in the presence of 100 mM NaCl (5, ○) taken with an inverse sign. (D) pH dependence of the absorption maximum in (1) WT (fit with pK_a values of 4.8 and 6.5), (2) D116N (pK_a value of 4.8), and (3) D251N (pK_a value of 5.1). Spectra were measured in 10 mM KCl. The pH was adjusted with HCl.

Figure 9

Properties of the D251E mutant in the initial state. (A) Shift of the absorption spectrum from an increase in pH at low salt concentrations (1–3 mM KCl): (1) pH 5.6, (2) pH 9.0, and (3) 10.6. (B) Different pH dependence of the absorption maximum of the D251E mutant (curves 1–3) and the WT (curves 4 and 5): (1) 100 mM KCl, (2) 100 mM NaCl, (3) 10 mM NaCl, (4) 100 mM NaCl, and (5) 100 mM KCl. (C) Absorption changes produced by (1) binding of H⁺ with a decrease in pH from 10.4 to 9.7 in 3 mM KCl, (2) addition of 10 mM NaCl to 3 mM KCl at pH 9.7, and (3) subsequent addition of 20 mM KCl (note that the latter change is the opposite of the others). (D) Red shift of the absorption maximum of the D251E mutant produced by the addition of NaCl at pH 10.3 (in the presence of 3 mM KCl). Such a shift does not occur in the WT under the same conditions.

Figure 10

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