Resonance Raman spectroscopy reveals the origin of an intermediate wavelength form in photoactive yellow protein.

Photoactive yellow protein (PYP) is a bacterial blue light receptor containing a 4-hydroxycinnamyl chromophore, and its absorption maximum is 446 nm. In a dark state, the hydroxyl group of the chromophore is deprotonated and forms hydrogen bonds with Tyr42 and Glu46. Either removal of a hydrogen bond with Tyr42 or addition of chaotropes such as thiocyanate produces a blue-shifted species called an intermediate wavelength form, in which absorption maximum ranges from 355 to 400 nm. To examine the structural origin of the intermediate wavelength form, we have performed resonance Raman investigations of wild-type PYP and some mutants (Tyr42 --> Ala, Tyr42 --> Phe, Glu46 --> Gln, and Thr50 --> Val) in the presence or absence of potassium thiocyanate. These studies show that the chromophore of the intermediate wavelength form is protonated, implying an increase in a pK(a) of the chromophore. Hence, the removal of the hydrogen bond between Tyr42 and chromophore or partial protein denaturation in the presence of thiocyanate results in a spectral blue-shift. Quantum chemical calculations based on density functional theory further support the idea that the pK(a) of the chromophore is increased by removing a hydrogen bond or by increasing the dielectric constant in the vicinity of the chromophore.