title: Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein.
abstract: Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands. ljgw.van.wilderen@few.vu.nl
authors.forename: L J G W
authors.initials: LJ
authors.lastname: van Wilderen
authors.forename: M A
authors.initials: MA
authors.lastname: van der Horst
authors.forename: I H M
authors.initials: IH
authors.lastname: van Stokkum
authors.forename: K J
authors.initials: KJ
authors.lastname: Hellingwerf
authors.forename: R
authors.initials: R
authors.lastname: van Grondelle
authors.forename: M L
authors.initials: ML
authors.lastname: Groot
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020607:Halorhodospira halophila / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D018462:Photobiology; D010777:Photochemistry; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000378:metabolism*; D013055:Spectrophotometry, Infrared
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria