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

Abstract

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±5nm, 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′ (∼2ms), 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 pKa of 6.4 and with I2′ the main species above this pKa. Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355nm at a delay of 20ms confirm this pKa value. I2 and I2′ are photoreversed with reversal times of ∼55μs and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pKa of ∼6.1. Photoreversal from I2′ dominates above the pKa. 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 pKa of ∼6.3. This equality of the equilibrium and kinetic pKa values is not accidental and suggests that k3 is proportional to [I2′].