Existence of two substates in the O intermediate of the bacteriorhodopsin photocycle

Published: 27 April 2022| Version 2 | DOI: 10.17632/y9xhs7hd3n.2
Tsutomu Kouyama


The excel file "BR-photoreaction-at various pHs-in-0MKCl-20220420.xlsx" contains raw data of flash-induced absorption changes in bacteriorhodopsin (bR) at various pH levels in low-salt membrane suspensions (~ 4 mM pH buffers) at 24 ℃. The excel file "BR-photoreaction-at various pHs-in-2MKCl-20220420.xlsx" contains raw data of flash-induced absorption changes in bacteriorhodopsin (bR) at various pH levels in high-salt membrane suspensions (2 M KCl) at 24 ℃. The excel file " BR-photoreaction-at various Temperatures-at-pH5.9-20220420b.xlsx" contains raw data of flash-induced absorption changes in bacteriorhodopsin (bR) at various temperatures in low-salt membrane suspensions (5 mM Mes) at pH 5.9. -------- ABSTRACT BR-photoreaction-at various pHs-in-0MKCl-2022elThe proton pumping cycle of bacteriorhodopsin (bR) is initiated when the retinal chromophore with telThe proton pumping cycle of bacteriorhodopsin (bR) is initiated when the retinal chromophore with the 13-trans configuration is photo-isomerized into the 13-cis configuration. To understand the recovery processes of the initial retinal configuration that occur in the late stage of the photocycle, we have performed a comprehensive analysis of absorption kinetics data collected at various pH levels and at different salt concentrations. The result of analysis revealed the following features of the late stages of the trans photocycle. i) Two substates occur in the O intermediate. ii) The visible absorption band of the first substate (O1) appears at a much shorter wavelength than that of the late substate (O2). iii) O1 is in rapid equilibrium with the preceding state (N), but O1 becomes less stable than N when an ionizable residue (X1) with a pKa value of 6.5 (in 2 M KCl) is deprotonated. iv) At a low pH and at a low salt concentration, the decay time constant of O2 is longer than those of the preceding states, but the relationship between these time constants is altered when the medium pH or the salt concentration is increased. On the basis of the present observations and previous studies on the structure of the chromophore in O, we suspect that the retinal chromophore in O1 takes on a distorted 13-cis configuration and the O1-to-O2 transition is accompanied by cis-to-trans isomerization about C13=C14 bond.


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Purple membrane was isolated from Halobacterium salinarum strain JW3 and purified according to established procedure and then stored at -80 ℃ in ~ 40 % sucrose (wt/wt). For the flash experiments, purple membrane was repeatedly washed with distilled water, and its concentration was adjusted to be ~ 0.8 OD at 568 nm in a 5 × 5 × 40-mm quartz cuvette. The medium pH of a membrane suspension was adjusted using a combination of buffer molecules (Mes, Pipes, Hepes, Taps, Ches, bicarbonate, citrate, glycine). Transient transmission data of bR were acquired using a computer-controlled experimental setup with a digital oscilloscope (LeCroy Wavesurfer 422) and a frequency-doubled Nd:YAG laser (Quantel Ultra, 3 ns, 532 nm)[39]. The flash intensity (~0.6 mJ/cm2) was adjusted such that the fraction of bR undergoing the proton pumping cycle was <10%. As the measuring light, emission from a 150 W Xe lamp was passed through a monochromator and directed to the sample cell, and the transmitted light was passed through a notch filter and the second monochromator and received by a photomultiplier tube connected to a transimpedance amplifier (Hamamatsu photonics, C13004-01). The notch filter was effective in reducing an undesired effect of the scattered component of light pulses. Flash-induced transmission changes were measured at 30 different wavelengths and at a wavelength interval of 10 nm. At each wavelength, the transmission signal was averaged 300-2000 times. After the systematic noise originating from the pulse-generating system was subtracted, the averaged transmission signal was rescaled to evaluate the time variation of absorbance. The absorption kinetics measured at various wavelengths, ΔA(λ,t), were analyzed using the singular-value decomposition (SVD) method. A semi-logarithm plot of the principal SVD components was investigated to determine the number (n) of exponents that were necessary to fit absorption kinetics data [41]). Then, the decay time constants of the exponents were refined by a global fitting. [The optimal value of n was subject to revise when two decay time constants were suspected to degenerate.] By plotting the amplitudes of the exponents against the wavelength of the measuring light, we obtained the transient difference spectra, 〖ΔA〗_i (λ) (i=1 ~ n): ΔA(λ,t)= ∑_(i=1)^n▒ 〖ΔA〗_i (λ) exp⁡(-k_i t) (1) To determine the absorption spectra of the reaction intermediates of bR, we employed a sequential irreversible model.


Nagoya Daigaku


Isomerization, Bacteriorhodopsin, Retinal Protein, Flash Photolysis, Kinetics, Biomembrane