Liquid-Microjet Photoelectron Spectroscopy of the Photoactive Yellow Protein Chromophore in Aqueous Solution

Photoactive yellow protein (PYP), a prototypical photoreceptor responsible for the photophobic response of the Halorhodospira halophila bacterium to harmful ultraviolet (UV) radiation, is known to undergo photooxidation in aqueous solution. However, the vertical detachment energy and electronic structure of the deprotonated chromophore that lies at the heart of PYP have not been measured in aqueous solution. Here, we use X-ray, extreme ultraviolet (EUV), and multiphoton UV liquid-microjet photoelectron spectroscopy, supported by high-level quantum chemistry calculations, to map out the electronic structure of the deprotonated PYP chromophore in aqueous solution. The vertical and adiabatic electron detachment energies are found to be 6.8 ± 0.1 eV and around 5.9 eV, respectively. Multiphoton UV photoelectron spectroscopy measurements confirm the existence of a high-lying two-photon resonance close to the detachment threshold that could be responsible for UV photooxidation, and they reveal the existence of a three-photon resonance in the detachment continuum. This work demonstrates the power of combining X-ray, EUV, and UV liquid-microjet photoelectron spectroscopy to unravel the electronic structure of weakly soluble organic chromophores, paving the way for deeper insights into their roles in photobiological processes.