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 ( I-1, I-1', and I-2') at pH 10. The spectrum of I-1' was obtained in two different ways. The maximal absorption is at 425 nm. I-1' probably has a deprotonated chromophore and may be regarded as the alkaline form of I-2'. At pH 10, the I-1 intermediate decays in 330 mu s in part to I-1' before I-1 and I-1' decay further to I-2' in similar to 1 ms. From the rise of I-2' ( similar to 1 ms) to the end of the photocycle, the three intermediates ( I-1, I-1', and I-2') remain in equilibrium and decay together to P in similar to 830 ms. Assuming that the spectra of I-1, I-1', and I-2' are pH-independent, their time courses were determined. On the millisecond to second time scale, they are in a pH-dependent equilibrium with a pK(a) of similar to 9.9. With an increase in pH, the I-1 and I-1' populations increase at the expense of the amount of I-2'. The apparent rate constant for the recovery of P slows with an increase in pH with a pK(a) of similar to 9.7. The equal pH dependence of this rate and the equilibrium concentrations follows, if we assume that the equilibration rates between the intermediates are much faster than the recovery rate and that the recovery occurs from I-2'. The pK(a) of similar to 9.9 is assigned to the deprotonation of the phenol of the surface-exposed chromophore in the I-1'-I-2' equilibrium. The I-1-I-1' equilibrium is pH-independent. Photoreversal experiments at pH 10 with the second flash at 355 nm indicate the presence of only one I-2-like intermediate, which we assign on the basis of its lambda(max) value to I-2'. After the rapid unresolved photoisomerization to I-2'(trans), the reversal pathway back to P involves two sequential steps ( 60 mu s and 3 ms). The amplitude spectra show that I-1'(trans) and I-1(trans) intermediates participate in this reversal.