Initial Steps of Signal Generation in Photoactive Yellow Protein Revealed with Femtosecond Mid-Infrared Spectroscopy

TLDR

Photoactive yellow protein is a bacterial blue‑light sensor that causes Halorhodospira halophila to swim away from intense blue light, and light absorption by its chromophore p‑coumaric acid initiates a photocycle with distinct intermediates. The study aims to describe the initial structural changes of the chromophore and adjacent amino acids. Visible pump/mid‑infrared probe spectroscopy is employed to monitor these changes. Photoexcitation bleaches the chromophore’s trans bands and shifts phenol ring bands, indicating charge translocation that likely drives trans‑to‑cis isomerization; breaking the hydrogen bond between the chromophore C=O and Cys69 and forming a stable cis ground state occur within ~2 ps, while hydrogen‑bond network rearrangements and coumaryl tail relaxation (0.9–1 ns) correspond to the I(0)→I(1) transition.

Abstract

Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's C=O group with amino acid Cys69 and formation of a stable cis ground state occur in approximately 2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromophore occurs in 0.9-1 ns, which event we identify with the I(0) to I(1) transition observed in visible spectroscopy.