Photoreaction of archaerhodopsin-2 at various pH levels
Description
The attached excel file "AR2-0MKCl-FlashPhotolysisData-20211228.xlsx" contains raw data of flash-induced absorption changes in archaerhodopsin-2 (aR2) at various pH levels in low-salt membrane suspensions (~4 mM pH buffers). The attached excel file "AR2-2MKCl-FlashPhotolysisData-20211227.xlsx" contains raw data of flash-induced absorption changes in archaerhodopsin-2 (aR2) at various pH levels in high-salt membrane suspensions (2 M KCl). --------------- Abstract The proton pumping cycle of archaerhodopsin-2 (aR2) was investigated over a wide pH range and at different salt concentrations. We have found that two substates, which are spectroscopically and kinetically distinguishable, occur in the O intermediate. The first O-intermediate (O1) absorbs maximumly at ~ 580 nm, whereas the late O-intermediate (O2) absorbs maximumly at 605 nm. At neutral pH, O1 is in rapid equilibrium with the N intermediate. When the medium pH is increased, O1 becomes less stable than N and, in proportion to the amount of O1 in the dynamic equilibrium between N and O1, the formation rate of O2 decreases. By contrast, the decay rate of O2 increases ~ 100 folds when the pH of a low-salt membrane suspension is increased from 5.5 to 7.5 or when the salt concentration is increased to 2 M KCl. Together with our recent study on two substrates in the O intermediate of bacteriorhodopsin (bR), the present study suggests that the thermally activated re-isomerization of the retinylidene chromophore into the initial all-trans configuration takes place in the O1-to-O2 transition; that is, O1 contains a distorted 13-cis chromophore. It is also found that the pKa value of the key ionizable residue (Asp101^aR2, Asp96^bR) in the proton uptake channel is elevated in the O1 state of aR2 as compared to the O1 state of bR. This mplies that the structural property of O1 in aR2 photocycle can be investigated over a wide pH range.
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Materials and Method: The claret membrane of Halorubrum Aus-2 was purified according to the established method for preparation of the purple membrane of Halobacterium salinarum. 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 aR2 at 24 °C were acquired using a computer-controlled experimental setup with a digital oscilloscope and a frequency-doubled Nd:YAG laser. The flash intensity (~0.6 mJ/cm2) was adjusted such that the fraction of aR2 undergoing the proton pumping cycle was <10%. The flash repetition rate (0.37 ~ 1.37 Hz) was adjusted such that the interval between flashes was much longer (> 5 times) than the turnover of the photocycle. 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 300-3000 times. The absorption kinetics measured at various wavelengths, ΔA(λ,t), were analyzed using the singular-value decomposition (SVD) method ((Lórenz-Fonfría & Heberle, 2014) and the number (n) of exponents that were necessary to fit absorption kinetics data were determined. After the decay time constants of the exponents were refined by a global fitting, the transient difference spectra, ΔA_i (λ) (i=1 ~ n), were evaluated: ΔA(λ,t)= ∑_i ΔA_i (λ) exp(-k_i t) (1) To determine the absorption spectra of the reaction intermediates of aR2, we employed a sequential irreversible model (Chizhov et al., 1996).