Biophysical journal

Kinetic and vibrational isotope effects of proton transfer reactions in channelrhodopsin-2.

PMID 26200864


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 1736xa0cm(-1) to 1738xa0cm(-1) in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8xa0cm(-1) in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9xa0cm(-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 20xa0ns to 5xa0ms is incompatible with early deprotonation of E90.