title: Mechanism of the AppABLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pKa on the Forward and Reverse Ground-State Reactions.
abstract: The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen-bonding network that surrounds the flavin chromophore on the nanosecond time scale, while the dark state of AppA is then recovered in a light-independent reaction with a dramatically longer half-life of 15 min. Residue Y21, a component of the hydrogen-bonding network, is known to be essential for photoactivity. Here, we directly explore the effect of the Y21 pKa on dark state recovery by replacing Y21 with fluorotyrosine analogues that increase the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements confirm that the structure of AppA is unperturbed by fluorotyrosine substitution, and that there is a small (3-fold) change in the photokinetics of the forward reaction over the fluorotyrosine series. However, reduction of 3.5 pH units in the pKa of Y21 increases the rate of dark state recovery by 4000-fold with a Brønsted coefficient of ∼ 1, indicating that the Y21 proton is completely transferred in the transition state leading from light to dark adapted AppA. A large solvent isotope effect of ∼ 6-8 is also observed on the rate of dark state recovery. These data establish that the acidity of Y21 is a crucial factor for stabilizing the light activated form of the protein, and have been used to propose a model for dark state recovery that will ultimately prove useful for tuning the properties of BLUF photosensors for optogenetic applications.
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Gil
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Haigney
authors.affiliation: School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: Sergey P
authors.initials: SP
authors.lastname: Laptenok
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Brust
authors.affiliation: School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: Andras
authors.initials: A
authors.lastname: Lukacs
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: James
authors.initials: J
authors.lastname: Iuliano
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Jeng
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Eduard
authors.initials: E
authors.lastname: Melief
authors.affiliation: School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: Rui-Kun
authors.initials: RK
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: EunBin
authors.initials: E
authors.lastname: Yoon
authors.affiliation: Central Laser Facility, Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Clark
authors.affiliation: Central Laser Facility, Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Towrie
authors.affiliation: Central Laser Facility, Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX, UK.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Greetham
authors.affiliation: William A. Shine Great Neck South High School, 341 Lakeville Rd, Great Neck, NY 11020, USA.
authors.forename: Annabelle
authors.initials: A
authors.lastname: Ng
authors.affiliation: William A. Shine Great Neck South High School, 341 Lakeville Rd, Great Neck, NY 11020, USA.
authors.forename: James
authors.initials: J
authors.lastname: Truglio
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.|Biochemistry & Cell Biology, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Jarrod
authors.initials: J
authors.lastname: French
authors.affiliation: School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Meech
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tonge
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry*; D005461:Fluorine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015394:Molecular Structure; D055668:Photochemical Processes; D011789:Quantum Theory; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D005461:Fluorine; D014443:Tyrosine