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  Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Lörinczi, E., Verhoefen, M.-K., Wachtveitl, J., Woerner, Glaubitz, C., Engelhard, M., et al. (2009). Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes. Journal of Molecular Biology, 393(2), 320-341. doi:10.1016/j.jmb.2009.07.055.

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 Creators:
Lörinczi, Eva1, Author           
Verhoefen, Mirka-Kristin2, Author
Wachtveitl, Josef2, Author
Woerner3, Author
Glaubitz, Clemens3, Author
Engelhard, Martin4, Author
Bamberg, Ernst1, Author           
Friedrich, Thomas1, 5, Author           
Affiliations:
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Institute of Physical and Theoretical Chemistry, Johann-Wolfgang-Goethe-University Frankfurt, 60438 Frankfurt am Main, Germany, ou_persistent22              
3Institute of Biophysical Chemistry, Johann-Wolfgang-Goethe-University Frankfurt, 60438 Frankfurt am Main, Germany, ou_persistent22              
4Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany, ou_persistent22              
5Institute of Chemistry, Sekr. PC-14, Technical University of Berlin, Straße des 17. Juni 135, D-10623 Berlin, Germany, ou_persistent22              

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Free keywords: proteorhodopsin; photocurrents; voltage dependence; vectoriality; Xenopus oocytes
 Abstract: Proteorhodopsin (PR), a light-driven proton pump from marine proteobacteria, exhibits photocycle characteristics similar to bacteriorhodopsin (BR) at neutral pH, including an M-like photointermediate. However, at acidic pH, spectroscopic evidence for an M-like species was absent, and the vectoriality of proton pumping was inverted. To gain further insight into this unusual property, we examined the voltage dependence of stationary and laser flash-induced photocurrents of PR under different pH conditions upon expression in Xenopus oocytes. The current–voltage curves were linear under all conditions tested, and photocurrent reversal potentials distinctly depended on the pH gradient. PR mutants D97N and D97T exhibited transient and stationary inward currents already at neutral pH, showing that neutralization of the proton acceptor abolishes forward pumping and permits only inward proton transport. Mutation E108G, which disrupts the donor site for Schiff base (SB) reprotonation, resulted in largely reduced photocurrents, which could be strongly stimulated by azide, similar to previous observations on BR mutant D96G. When PR and BR photocurrents in response to blue or green laser flashes during or after continuous illumination were compared, direct electrical evidence for the occurrence of an M-like intermediate at neutral pH could only be obtained when reprotonation of the SB was slowed down by PR mutation E108G. For PR at acidic pH, laser flashes only produced inwardly directed photocurrents, independent from background illumination, thus precluding electrical identification of an M-like species. However, when visible absorption spectroscopy was carried out at low temperatures, occurrence of an M-like species was robustly observed at low pH. This indicates that SB deprotonation and reprotonation occur during the PR photocycle also at low pH. Our results corroborate the conclusion that in PR, the direction of proton pumping can be switched by changes in pH and membrane potential, with the protonation state of Asp-97 being the key determinant for selecting between transport modes.

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Language(s): eng - English
 Dates: 2009-06-152009-03-132009-07-172009-07-232009-10-23
 Publication Status: Published in print
 Pages: 22
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.jmb.2009.07.055
PMID: 19631661
 Degree: -

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Title: Journal of Molecular Biology
  Other : JMB
  Abbreviation : J. Mol. Biol.
Source Genre: Journal
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Publ. Info: Elsevier
Pages: - Volume / Issue: 393 (2) Sequence Number: - Start / End Page: 320 - 341 Identifier: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/0022-2836