Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.
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.
Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.
Sergei P
SP
Balashov
Eleonora S
ES
Imasheva
Andrei K
AK
Dioumaev
Jennifer M
JM
Wang
Kwang-Hwan
KH
Jung
Janos K
JK
Lanyi
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D001665:Binding Sites; D041965:Flavobacteriaceae / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055668:Photochemical Processes; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D025604:Rhodopsins, Microbial / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D012545:Schiff Bases / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D025604:Rhodopsins, Microbial; D012545:Schiff Bases; D001224:Aspartic Acid; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase