Direct observation of the pH-dependent equilibrium between L-like and M intermediates of photoactive yellow protein.

Equilibrium between the photoproducts of photoactive yellow protein (PYP), present in a millisecond time scale, was studied. The near-UV intermediate of PYP (PYPM) was red-shifted by alkalization due to the deprotonation of the chromophore (pKa=10.2). In addition, a small amount of red-shifted intermediate coexisted with PYPM. Its spectral shape in the visible region agreed with that of PYPL, the precursor of PYPM. The fraction of PYPL-like product was maximal at pH 10. It decays with a rate constant identical to that of PYPM. These results indicate that PYPL-like product is in pH-dependent equilibrium with PYPM and deprotonated PYPM.