Photocycle and Photoreversal of Photoactive Yellow Protein at Alkaline pH: Kinetics, Intermediates, and Equilibria†

Since the habitat of Halorhodospira halophila is distinctly alkaline, we investigated the kinetics and intermediates of the photocycle and photoreversal of the photoreceptor photoactive yellow protein (PYP) from pH 8 to 11. SVD analysis of the transient absorption time traces in a broad wavelength range (330−510 nm) shows the presence of three spectrally distinct species (I1, I1‘, and I2‘) at pH 10. The spectrum of I1‘ was obtained in two different ways. The maximal absorption is at 425 nm. I1‘ probably has a deprotonated chromophore and may be regarded as the alkaline form of I2‘. At pH 10, the I1 intermediate decays in ∼330 μs in part to I1‘ before I1 and I1‘ decay further to I2‘ in ∼1 ms. From the rise of I2‘ (∼1 ms) to the end of the photocycle, the three intermediates (I1, I1‘, and I2‘) remain in equilibrium and decay together to P in ∼830 ms. Assuming that the spectra of I1, I1‘, and I2‘ are pH-independent, their time courses were determined. On the millisecond to second time scale, they are in a pH...