Low-temperature FTIR study of multiple K intermediates in the photocycles of bacteriorhodopsin and xanthorhodopsin

Abstract

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.

Figure 1

Low-temperature FTIR spectra of bacteriorhodopsin (panel A) and xanthorhodopsin (panel B) in H₂O (red) and D₂O (blue), trapped during blue illumination at 80K.

Figure 2

Light-induced mixtures of K states trapped during blue-illumination at different temperatures. In bacteriorhodopsin (panels A and B) and in xanthorhodopsin (panels C and D) at 80K (red), 100K (yellow), 110K (green), 120K (cyan), 130K (blue) and 140K (magenta) for bacteriorhodopsin and at 80K (red), 110K (yellow), 130K (green), 150K (cyan), 170K (blue) and 190K (magenta) for xanthorhodopsin, respectively.

Figure 3

Decomposition of the region of the HOOP bands (from Figure 2) with a weighted sum of Gaussian and Lorentzian components: in bacteriorhodopsin spectra at 80 and 130K (panel A and B), and in xanthorhodopsin spectra at 80, 130 and 190K (panel C-E). The deconvolution was done in GRAMS software, yielding fits with r² better than 0.997. The fit residuals are plotted in green. In bacteriorhodopsin the positions (their widths in brackets) for the fitted bands averaged over six temperatures are at: 941.6±0.4 cm⁻¹ (5.1±0.6), 956.2±0.1 cm⁻¹ (6.9±0.5), 962.9±0.4 cm⁻¹ (5±1), 974.8±0.3 cm⁻¹ (6.6±0.6), 985±2 cm⁻¹ (15±1), 997.0±0.3 cm⁻¹ (4.7±0.5), 1008.0±0.1 cm⁻¹ (2.8±0.3). In xanthorhodopsin the corresponding values are: 941.5±0.3 cm⁻¹ (5.2±0.7), 953.4±0.6 cm⁻¹ (7.5±0.8), 960.0±0.2 cm⁻¹ (6.8±0.6), 971.5±0.2 cm⁻¹ (8±2), 980.5±0.6 cm⁻¹ (12±1), 992±2 cm⁻¹ (8±2), 999.0±0.7 cm⁻¹ (5±1), 1005.6±0.2 cm⁻¹ (4.1±0.3). The two main temperature-sensitive bands are at 956 cm⁻¹ (in blue) and 985 cm⁻¹ (in red) in bacteriorhodopsin (panel A-B), and at 960 cm⁻¹ (in blue) and 981 cm⁻¹ (in red) in xanthorhodopsin (panel C-E). Previously, the Raman band at 1008 cm⁻¹ in non-excited BR₅₆₈ was assigned to the symmetric methyl rock, i.e., to the in-plane rather than out-of-plane vibration, implying a similar assignment for the negative band (i.e. depletion in non-excited state) at 1008 cm⁻¹ in bacteriorhodopsin and two similar bands, at ~1000 and at ~1005 cm⁻¹, in xanthorhodopsin.

Figure 4

Temperature dependence of the HOOP bands in xanthorhodopsin. Panel A: The amplitude of the decomposed components (as in Figure 3). Panel B: the ratio of amplitudes of the two main temperature-dependent bands A₉₆₀/A₉₈₁.

Figure 5

Temperature perturbation experiments with bacteriorhodopsin. Panel A: Effect of up-shift of temperature, which illustrates the forward reaction; the light-induced conversion was initiated at 80K (in blue) and the sample was later heated to 140K (in red) in the dark. Panel B: Absence of effect from down-shift of temperature, which illustrates the absence of the back reaction; the light-induced conversion was initiated at 140K (in red) and the sample was later cooled to 80K (in red) in the dark. The shoulder in the ethylenic band reflects incomplete light-adaptation, most probably due to insufficient hydration of the sample.

Figure 6

Two distinct K intermediates in bacteriorhodopsin (panel A) and xanthorhodopsin (panel B) obtained from the spectra in Figure 2 by decomposition, involving SVD, rotation, and subtraction (see text for details).

Figure 7

Fractional contributions for the two K states calculated using the deconvoluted spectra of the pure forms (Figure 6) and the raw spectra in Figure 2 (see text for details).

Figure 8

The spectra of the two K-like states in the water region in bacteriorhodopsin (panel A and B) and in xanthorhodopsin (panel C and D). The spectra in panels A and B are at 2 cm⁻¹, as all the rest data in this paper; for presentation purposes the spectra in panels C and D were additionally smoothed to ~3.5 cm⁻¹ resolution. Panels A and C are the measured IR spectra (corresponding to the spectra in Figure 2) of mixtures of the two K-like states trapped during blue light illumination at particular temperatures. Panel A: in bacteriorhodopsin at 80 (red), 100 (yellow), 110 (green), 120 (cyan), 130 (blue) and 140K (magenta). Panel B: in xanthorhodopsin at 85K (red) and 180K (blue). Panel B and D: Spectral decomposition based on the relative contributions (Figure 7) of the two states calculated from spectra in Figure 6 (dashed lines) and their additional adjustment (solid lines). See text for details.