Low-temperature FTIR study of multiple K intermediates in the photocycles of bacteriorhodopsin and xanthorhodopsin.
Low-temperature FTIR spectroscopy of bacteriorhodopsin and xanthorhodopsin was used to elucidate the number of K-like bathochromic states, their sequence, and their contributions to the photoequilibrium mixtures created by illumination at 80-180 K. We conclude that in bacteriorhodopsin the photocycle includes three distinct K-like states in the sequence bR (hv)--> I* --> J --> K(0) --> K(E) --> L --> ..., and similarly in xanthorhodopsin. K(0) is the main fraction in the mixture at 77 K that is formed from J. K(0) becomes thermally unstable above approximately 50 K in both proteins. At 77 K, both J-to-K(0) and K(0)-to-K(E) transitions occur and, contrarily to long-standing belief, cryogenic trapping at 77 K does not produce a pure K state but a mixture of the two states, K(0) and K(E), with contributions from K(E) of approximately 15 and approximately 10% in the two retinal proteins, respectively. Raising the temperature leads to increasing conversion of K(0) to K(E), and the two states coexist (without contamination from non-K-like states) in the 80-140 K range in bacteriorhodopsin, and in the 80-190 K range in xanthorhodopsin. Temperature perturbation experiments in these regions of coexistence revealed that, in spite of the observation of apparently stable mixtures of K(0) and K(E), the two states are not in thermally controlled equilibrium. The K(0)-to-K(E) transition is unidirectional, and the partial transformation to K(E) is due to distributed kinetics, which governs the photocycle dynamics at temperatures below approximately 245 K (Dioumaev and Lanyi, Biochemistry 2008, 47, 11125-11133). From spectral deconvolution, we conclude that the K(E) state, which is increasingly present at higher temperatures, is the same intermediate that is detected by time-resolved FTIR prior to its decay, on a time scale of hundreds of nanoseconds at ambient temperature (Dioumaev and Braiman, J. Phys. Chem. B 1997, 101, 1655-1662), into the K(L) state. We were unable to trap the latter separately from K(E) at low temperature, due to the slow distributed kinetics and the increasingly faster overlapping formation of the L state. Formation of the two consecutive K-like states in both proteins is accompanied by distortion of two different weakly bound water molecules: one in K(0), the other in K(E). The first, well-documented in bacteriorhodopsin at 77 K where K(0) dominates, was assigned to water 401 in bacteriorhodopsin. The other water molecule, whose participation has not been described previously, is disturbed on the next step of the photocycle, in K(E), in both proteins. In bacteriorhodopsin, the most likely candidate is water 407. However, unlike bacteriorhodopsin, the crystal structure of xanthorhodopsin lacks homologous weakly bound water molecules.
Department of Physiology & Biophysics, University of California, Irvine, California 92697, USA. 5R37GM029498),
Andrei K
AK
Dioumaev
Jennifer M
JM
Wang
Janos K
JK
Lanyi
D001426:Bacterial Proteins / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry*; D010777:Photochemistry; D025604:Rhodopsins, Microbial / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D001426:Bacterial Proteins; D025604:Rhodopsins, Microbial; C519152:xanthorhodopsin, Salinibacter; D001436:Bacteriorhodopsins