pH Dependence of the Photocycle Kinetics of the E46Q Mutant of Photoactive Yellow Protein: Protonation Equilibrium between I1 and I2 Intermediates, Chromophore Deprotonation by Hydroxyl Uptake, and Protonation Relaxation of the Dark State†

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I2 intermediate. For E46Q we find that I2 is in a pH-dependent equilibrium with its precursor I1‘ with a pKa of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I1‘ to I2 transition one proton is taken up from the external medium. The pKa of 8.15 is that of the surface-exposed chromophore in the equilibrium between I1‘ and I2 and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I1‘/I2 equilibrium with associated H+ uptake is reminiscent of the MI/MII equilibrium in the formation of the signaling state of rhodopsin. Well above this pKa no I2 is formed and I1‘ returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I2 is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (fir...