title: Low temperature FTIR spectroscopy provides new insights in the pH-dependent proton pathway of proteorhodopsin.
abstract: In the presented study the low pH photocycle of proteorhodopsin is extensively investigated by means of low temperature FTIR spectroscopy. Besides the already well-known characteristics of the all-trans and 13-cis retinal vibrations the 77K difference spectrum at pH 5.1 shows an additional negative signal at 1744 cm(-1) which is interpreted as indicator for the L state. The subsequent photocycle steps are investigated at temperatures higher than 200K. The combination of visible and FTIR spectroscopy enabled us to observe that the deprotonation of the Schiff base is linked to the protonation of an Asp or Glu side chain - the new proton acceptor under acidic conditions. The difference spectra of the late intermediates are characterized by large amide I changes and two further bands ((-)1751 cm(-1)/(+)1725 cm(-1)) in the spectral region of the Asp/Glu ν(C=O) vibrations. The band position of the negative signature points to a transient deprotonation of Asp-97. In addition, the pH dependence of the acidic photocycle was investigated. The difference spectra at pH 5.5 show distinct differences connected to changes in the protonation state of key residues. Based on our data we propose a three-state model that explains the complex pH dependence of PR.
authors.affiliation: Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue Straße 7, 60438 Frankfurt am Main, Germany.
authors.forename: Mirka-Kristin
authors.initials: MK
authors.lastname: Verhoefen
authors.forename: Gabriela
authors.initials: G
authors.lastname: Schäfer
authors.forename: Sarika
authors.initials: S
authors.lastname: Shastri
authors.forename: Ingrid
authors.initials: I
authors.lastname: Weber
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
authors.forename: Werner
authors.initials: W
authors.lastname: Mäntele
authors.forename: Josef
authors.initials: J
authors.lastname: Wachtveitl
mesh_terms: D001426:Bacterial Proteins; D003080:Cold Temperature; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008027:Light; D008958:Models, Molecular; D011487:Protein Conformation; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry*; D025604:Rhodopsins, Microbial; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D011522:Protons; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin