Photoreaction kinetics of archaerhodopsin-2 at various pH levels
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.
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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. 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 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 method and and the number (n) of exponents that were necessary to fit absorption kinetics data were determined. Then, the decay time constants of the exponents were refined by a global fitting. 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 ΔA_i (λ) exp(-k_i t) (1) Next, we determined the absorption spectra of the reaction intermediates of bR by employing a sequential irreversible model.