The transient accumulation of the signaling state of photoactive yellow protein is controlled by the external pH

The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I-2'. The pH dependence of the equilibrium between the transient photocycle intermediates I-2 and I-2' was investigated. The formation of I-2' from I-2 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: I-1, I-2, and I-2'. Their spectra were determined using the extrapolated difference method. I-2 and I-2' 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 I-1, I-2, and I-2' intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I-2' (similar to 2 ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I-2 and I-2' is pH dependent with a pKa of 6.4 and with I-2' the main species above this pK(a). Measurements of the pH dependence of the photoreversal kinetics with a second. ash of 355 nm at a delay of 20 ms confirm this pK(a) value. I-2 and I-2' are photoreversed with reversal times of similar to 55 mu s and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pK(a) of similar to 6.1. Photoreversal from I-2' dominates above the pK(a). The transient accumulation of I-2', the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k(3) for the recovery to the initial dark state also has a pK(a) of similar to 6.3. This equality of the equilibrium and kinetic pKa values is not accidental and suggests that k3 is proportional to [I-2'].