title: Kinetic and vibrational isotope effects of proton transfer reactions in channelrhodopsin-2.
abstract: Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm(-1) to 1738 cm(-1) in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm(-1) in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm(-1) and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin, Berlin, Germany.
authors.forename: Tom
authors.initials: T
authors.lastname: Resler
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin, Berlin, Germany.
authors.forename: Bernd-Joachim
authors.initials: BJ
authors.lastname: Schultz
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin, Berlin, Germany.
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz-Fonfría
authors.affiliation: Genetic Biophysics at Department of Physics, Freie Universität Berlin, Berlin, Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Schlesinger
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin, Berlin, Germany. Electronic address: joachim.heberle@fu-berlin.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii; D017666:Deuterium Oxide / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D010782:Photolysis; D010843:Pichia; D011522:Protons; D013057:Spectrum Analysis; D014732:Vibration; D014867:Water / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D011522:Protons; C523009:channelopsin-2, Chlamydomonas reinhardtii; D014867:Water; D017666:Deuterium Oxide