Resonance Raman evidence for two conformations involved in the L intermediate of photoactive yellow protein

The blue light receptor photoactive yellow protein (PYP) displays a photocycle that involves several intermediate states. Here we report resonance Raman spectroscopic investigations of the short-lived red-shifted intermediate denoted PYPL. We have found that the Raman bands of the carbonyl C = O stretching mode nu(11) as well as the C = C stretching mode nu(13) for the chromophore can be resolved into two peaks, and the ratio of the two components varies as a function of pH with pK(a) similar to6. The isotope effects on the resonance Raman spectra have confirmed a deprotonated cis-chromophore for the two components. The results indicate the presence of two conformations in the active site of PYPL. The normal coordinate calculations based on the density functional theory provide a structural model for the two conformations, where the low pH form is possibly an active structure for the protonation reaction generating a following intermediate in the photocycle.