Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

Abstract

The time course of thermal reactions after illumination of 100% humidified bacteriorhodopsin films was followed with FTIR spectroscopy between 125 and 195 K. We monitored the conversion of the initial photoproduct, K, to the next, L intermediate, and a shunt reaction of the L state directly back to the initial BR state. Both reactions can be described by either multiexponential kinetics, which would lead to apparent end-state mixtures that contain increasing amounts of the product, i.e., L or BR, with increasing temperature, or distributed kinetics. Conventional kinetic schemes that could account for the partial conversion require reversible reactions, branching, or parallel cycles. These possibilities were tested by producing K or L and monitoring their interconversion at a single temperature and by shifting the temperature upward or downward after an initial incubation and after their redistribution. The results are inconsistent with any conventional scheme. Instead, we attribute the partial conversions to the other alternative, distributed kinetics, observed previously in myoglobin, which arise from an ensemble of frozen conformational substates at the cryogenic temperatures. In this case, the time course of the reactions reflects the progressive depletion of distinct microscopic substates in the order of their increasing activation barriers, with a distribution width for K to L reaction of approximately 7 kJ/mol.

Fig. 1.

FTIR difference spectra of bacteriorhodopsin films at different temperatures, during and after illumination. Spectra during illumination are shown in red, spectra after 200 min of subsequent thermal reaction are in blue. (a) Spectra at 125 K (blue-light illumination). (b) Spectra at 155 K (blue-light illumination). (c) Spectra at 195 K (red-light illumination).

Fig. 2.

FTIR difference spectra. (a) K_LT was measured at 100 K; spectrum is shown in red. Calculated K is shown in blue. (b) Calculated L. The latter two spectra were calculated from data as in Fig. 1b measured at 135, 145, and 155 K. At each temperature, a spectrum under illumination (e.g., Fig. 1b, red) was subtracted from the spectrum at ≈200 min (e.g., Fig. 1b) to eliminate K-specific (26) spectral features. The resulting pure but unscaled spectrum for L was subtracted from the spectrum under illumination to eliminate L-specific spectral features (26), producing a pure but unscaled K spectrum. The coefficients of these two subtractions were used for scaling K and L at 135, 145, and 155 K, which were then averaged.

Fig. 3.

Time course of IR features from K (ethylenic stretch band), shown on a logarithmic scale vs. linear time (a) and vs. logarithmic time (b) at various temperatures, after a photostationary state containing predominantly K was created at the same temperature with blue light. (a) Fitted curves are to multicomponent exponentials. (b) For clarity, only every third data point is displayed. Fitted curves are to the empirical equation, rate = (1+ t/t_o)⁻ⁿ (6).

Fig. 4.

Time course of the decay of IR features from L (CO stretch band), illustrating the decay of L to BR (Fig. 1c) at various temperatures, after a photostationary state containing virtually only L was created at the same temperature with red light. The data and the fitted curves are as in Fig. 3 a and b. (b) For clarity, only every third data point is displayed.

Fig. 5.

Limiting amounts of the L state formed from K (open circles) and the recovered BR state from L (closed circles) that accumulate after prolonged incubation at various temperatures, as in Figs. 3 and 4. At temperatures above 165 K, the decay of the L state to BR (Fig. 4) would interfere with further monitoring of the K → L reaction. Likewise, the recovery of the BR state from L could not be determined at above 195 K because of interference by the L → M reaction. Therefore, the sigmoidal curves were calculated only from data points below 165 and 195 K for the two curves, respectively, but for clarity the lines are drawn as if at higher temperatures the L→ BR and L→ M reactions did not interfere.

Fig. 6.

Tests of the reversibility of the K → L reaction. Filled circles represent the time course when the K state was created by blue illumination and its decay was followed at either 135 or 155 K (as in Fig. 3a). Open circles are data from separate experiments, with K created and its dynamics monitored for the first 180 min at 135 K, followed by shift of the temperature to 155 K (a) or created and monitored for the first 140 min at155 K, and then the temperature was shifted to 135 K (b). In both cases, the time course of the temperature-induced changes was monitored after the temperature jump, but no data were collected during the reequilibration of the temperature, for periods of 130 min (a) and 40 min (b). (a) The dashed line is the estimate of the further progress of the K → L reaction at the higher temperature. (b) The dashed line illustrates what would be expected (approximately) if the L → K back-reaction occurred.

Fig. 7.

Calculation of activation energy spectra for the K → L reaction from the fits in Fig. 3b, as described in ref. and the text. (a) Test of the applicability of the method in ref. and estimation of the activation energy at the peak of the distribution curve and the preexponential factor. The mean activation enthalpy is 41 kJ/mol, and the preexponential factor, A, is ≈10⁻¹¹ s⁻¹. (b) Calculated energy spectrum. The spectrum in bold is the average of the curves calculated at 135, 140, 145, and 155 K; the shaded area corresponds to ±1 SD. The full-width at half-maximum of the distribution function is ≈7 kJ/mol.