Time-resolved FTIR study of light-driven sodium pump rhodopsins

Abstract

Light-driven sodium ion pump rhodopsin (NaR) is a new functional class of microbial rhodopsin. A previous flash photolysis study of Krokinobacter eikastus rhodopsin 2 (KR2) revealed the presence of three kinetically distinct intermediates: K, L/M, and O. Previous low-temperature Fourier-transform infrared (FTIR) spectroscopy of KR2 showed that photoisomerization from the all-trans to the 13-cis form is the primary event of the Na+ pumping photocycle, but structural information on the subsequent intermediates is limited. Here, we applied step-scan time-resolved FTIR spectroscopy to KR2 and Nonlabens dokdonensis rhodopsin 2 (NdR2). Both low-temperature static and time-resolved FTIR spectra resolved a K-like intermediate, and the corresponding spectra showed few differences. Strong hydrogen-out-of-plane (HOOP) vibrations, which appeared in the K intermediate, are common among other rhodopsins. It is, however, unique for NaR that such HOOP bands are persistent in late intermediates, such as L and O intermediates. This observation strongly suggests similar chromophore structures for the K, L, and O intermediates. In fact, an isotope-labeled study that used 12,14-D2 retinal revealed that the chromophore configuration of the O intermediate in NaR is 13-cis. In contrast to the vibrations of the chromophore, those of the protein differ among intermediates, and this is related to the sodium-pumping function. The molecular mechanism of the light-driven sodium pump is discussed on the basis of the present time-resolved FTIR results.

Molecular mechanism of light-driven sodium pumping

“…3). Surprisingly, all-trans configuration of the retinal in the O-state is in contrast to the recently published timeresolved Fourier-transform infrared spectroscopy (FTIR) data, where authors suggested 13-cis configuration in the O-state of NaRs 21 , but is in line with the data on another light-driven Na + pump from Gillisia limnaea (GLR), published in 2014, where the authors report a distorted all-trans configuration of retinal in the O-state 22 . In ref.…”

“…This distortion may result in the appearing of the peak at 940 cm −1 described in ref. 21 . We also cannot exclude that the inconsistency of the results on the retinal configuration in the O-state may originate from the different conditions and protein environment during the experiments.…”

Femtosecond-to-millisecond structural changes in a light-driven sodium pump

“…Most importantly in terms of function, KR2 in treated crystals follows a photocycle (Fig. 1) identical to that observed by our absorption spectroscopy on purified KR2 in agreement with previous reports 19,18 .…”

“…The pumping cycle of KR2 has been studied by a variety of time-resolved spectroscopic techniques 1,[13][14][15][16][17][18][19] . High-resolution structures of the resting state have been determined in various forms by X-ray crystallography 2,3,20 .…”

Temperature Dependence of theKrokinobacterrhodopsin 2 Kinetics

“…While at first, it was modeled as an unbranched unidirectional photocycle with five distinct photointermediates, 14 later on more complex models were proposed. 15,16,17 KR2's photocycle has already been studied extensively with various techniques [18][19][20][21][22][23] but still remains to be understood completely. Especially the spectral location of the photointermediates, as well as the equilibria within the photocycle remain elusive.…”