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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.

SUBMITTER: Gil A

PROVIDER: S-EPMC4830125 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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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.

Gil Agnieszka A Haigney Allison A Laptenok Sergey P SP Brust Richard R Lukacs Andras A Iuliano James J Jeng Jessica J Melief Eduard E Zhao Rui-Kun RK Yoon EunBin E Clark Ian I Towrie Michael M Greetham Gregory M GM Ng Annabelle A Truglio James J French Jarrod J Meech Stephen R SR Tonge Peter J PJ

Journal of the American Chemical Society 20160115 3

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 ...[more]

PMID: 26708408

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Project description:The forward and reverse H + CO ↔ HCO reactions are important for combustion chemistry and have been studied from a wide variety of theoretical and experimental techniques. However, most of the chemical kinetic investigations concerning these processes are focused on low pressures or fall-off regions. Hence, a high-level electronic structure treatment was employed here in order to provide accurate rate constant values for these reactions at the high-pressure limit along temperatures from 50 to 4000 K. In relative terms, the variational effects on rate constants are shown to be almost as important at high temperatures as quantum tunneling corrections at the lowest temperatures investigated. The activation energies fitted by using modified and traditional Arrhenius' equations for the forward rate constants from 298 to 2000 K are, respectively, equal to 2.64 and 3.89 kcal mol-1, while similar fittings provided, respectively, 1.96 and 3.22 kcal mol-1, considering now forward rate constants from a temperature range of 298-373 K. This last activation energy determination (3.22 kcal mol-1) is in better agreement with the commonly referenced experimental value of 2.0 ± 0.4 kcal mol-1, also obtained from traditional fittings in the range 298-373 K, than the value attained from a broader temperature range fitting (3.89 kcal mol-1). However, some additional care must be considered along these comparisons once the experimental reaction rate measurements have been done for the trimolecular H + CO + M → HCO + M reaction instead. Anyway, the usage of appropriate temperature ranges is fundamental for reliable activation energy comparisons, although the remaining deviation between theory and experiment is still large and is possibly caused by the different pressure regimes assessed in each case. Finally, we roughly estimated that the high-pressure limit for the HCO decomposition into H and CO can be achieved along temperatures ranging from ∼246 and ∼255 K downward, respectively, at pressures of 1.1 and 9.6 atm, although further experiments should be carried out in order to improve these estimates. On the other hand, pressures larger than 9.8 × 104 atm are required for the aforementioned dissociation reaction to attain the high-pressure limit at 700 K. Therefore, the rate constants determined here are probably applicable in combustion studies at lower temperatures.

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Dataset Information

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.

Publications

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.

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