We report the isolation, functional reconstitution and photophysical characterisation of Rhodobacter sphaeroides photoactive yellow protein (PYP), of which the gene was recently cloned. Reconstitution of the his-tagged purified apo-protein with 4-hydroxy-cinnamic acid yields the characteristic blue absorbance at 446 nm, but surprisingly also an absorbance peak at 360 nm. This additional peak is not caused by binding of a second chromophore, as confirmed with mass spectroscopy. Moreover, reconstitution with the ‘locked’ analogue 7-hydroxy-coumarin-3-carboxylic acid yields only a single absorbance peak at 441 nm. The 446 nm and 360 nm species are part of a temperature- and pH-dependent equilibrium. Photoactivation of the protein leads to formation of a blue-shifted intermediate as in other PYPs, with a 100-fold increased groundstate recovery rate (k pB→pG=500 s−1) compared to E-PYP.
