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The Transient Accumulation of the Signaling State of Photoactive Yellow Protein Is Controlled by the External pH

Berthold Borucki ^*, Chandra P. Joshi ^*, Harald Otto ^*, Michael A. Cusanovich  and Maarten P. Heyn ^*

^* Biophysics Group, Department of Physics, Freie Universität Berlin, 14195 Berlin, Germany; and Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona, 85721

Correspondence: Address reprint requests to Maarten P. Heyn, Biophysics Group, Dept. of Physics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany. Tel: 49-30-83856160; Fax: 49-30-83856299; E-mail: heyn{at}physik.fu-berlin.de.

The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I₂'. The pH dependence of the equilibrium between the transient photocycle intermediates I₂ and I₂' was investigated. The formation of I₂' from I₂ is accompanied by a major conformational change. The kinetics and intermediates of the photocycle and of the photoreversal were measured by transient absorption spectroscopy from pH 4.6 to 8.4. Singular value decomposition (SVD) analysis of the data at pH 7 showed the presence of three spectrally distinguishable species: I₁, I₂, and I₂'. Their spectra were determined using the extrapolated difference method. I₂ and I₂' have electronic absorption spectra, with maxima at 370 ± 5 and 350 ± 5 nm, respectively. Formation of the signaling state is thus associated with a change in the environment of the protonated chromophore. The time courses of the I₁, I₂, and I₂' intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I₂' (2 ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I₂ and I₂' is pH dependent with a pK_a of 6.4 and with I₂' the main species above this pK_a. Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355 nm at a delay of 20 ms confirm this pK_a value. I₂ and I₂' are photoreversed with reversal times of 55 µs and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pK_a of 6.1. Photoreversal from I₂' dominates above the pK_a. The transient accumulation of I₂', the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k₃ for the recovery to the initial dark state also has a pK_a of 6.3. This equality of the equilibrium and kinetic pK_a values is not accidental and suggests that k₃ is proportional to [I₂'].

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