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Direct determination of resonance energy transfer in photolyase: structural alignment for the functional state.

Tan C, Guo L, Ai Y, Li J, Wang L, Sancar A, Luo Y, Zhong D - J Phys Chem A (2014)

Bottom Line: Photoantenna is essential to energy transduction in photoinduced biological machinery.A photoenzyme, photolyase, has a light-harvesting pigment of methenyltetrahydrofolate (MTHF) that transfers its excitation energy to the catalytic flavin cofactor FADH¯ to enhance DNA-repair efficiency.We observed 170 ps for excitation energy transferring to the fully reduced hydroquinone FADH¯, 20 ps to the fully oxidized FAD, and 18 ps to the neutral semiquinone FADH(•), and the corresponding orientation factors (κ(2)) were determined to be 2.84, 1.53 and 1.26, respectively, perfectly matching with our calculated theoretical values.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Department of Chemistry and Biochemistry, and Programs of Biophysics, Chemical Physics, and Biochemistry, The Ohio State University , 191 West Woodruff Avenue, Columbus, Ohio 43210, United States.

ABSTRACT
Photoantenna is essential to energy transduction in photoinduced biological machinery. A photoenzyme, photolyase, has a light-harvesting pigment of methenyltetrahydrofolate (MTHF) that transfers its excitation energy to the catalytic flavin cofactor FADH¯ to enhance DNA-repair efficiency. Here we report our systematic characterization and direct determination of the ultrafast dynamics of resonance energy transfer from excited MTHF to three flavin redox states in E. coli photolyase by capturing the intermediates formed through the energy transfer and thus excluding the electron-transfer quenching pathway. We observed 170 ps for excitation energy transferring to the fully reduced hydroquinone FADH¯, 20 ps to the fully oxidized FAD, and 18 ps to the neutral semiquinone FADH(•), and the corresponding orientation factors (κ(2)) were determined to be 2.84, 1.53 and 1.26, respectively, perfectly matching with our calculated theoretical values. Thus, under physiological conditions and over the course of evolution, photolyase has adopted the optimized orientation of its photopigment to efficiently convert solar energy for repair of damaged DNA.

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Femtosecond-resolved transient-absorption dynamics probedfrom500 to 700 nm upon excitation at 400 nm. The transients can be systematicallydeconvoluted to three contributions (insets A–D) of the excitedMTHF (dashed blue), the component resulting from the excited FAD fromRET of MTHF* (dashed dark goldenrod), and the signal from the directlyexcited FAD (dashed pink). Note the distinct rise signals reflectingthe intermediate formation through RET of MTHF* and the signal ofthe excited FAD containing its photoreduction dynamics; see the text.
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fig5: Femtosecond-resolved transient-absorption dynamics probedfrom500 to 700 nm upon excitation at 400 nm. The transients can be systematicallydeconvoluted to three contributions (insets A–D) of the excitedMTHF (dashed blue), the component resulting from the excited FAD fromRET of MTHF* (dashed dark goldenrod), and the signal from the directlyexcited FAD (dashed pink). Note the distinct rise signals reflectingthe intermediate formation through RET of MTHF* and the signal ofthe excited FAD containing its photoreduction dynamics; see the text.

Mentions: Figure 5 shows the absorption transients probedfrom 500to 700 nm. Similarly, we observed a distinct rise in tens of picosecondsfrom 580 to 640 nm (also see Figure 5B,C),indicating an intermediate (FAD*) formation. Similar to the analysesabove for the FADH¯ state, all signals are well fit by threeparts: The first one represents the decay of MTHF* absorption or stimulatedemission (negative formation signals in Figure 5B,C) with a time constant of 20 ps determined from the fluorescencequenching dynamics, the second signal results from the directly excitedFAD* species at 400 nm, and the third part is related to the transferredFAD* from MTHF* with a distinct rise and decay pattern. It shouldbe noted that the signal even from the directly excited FAD* at 400nm in Figure 5 is complex and contains manycomponents of different species. We have systematically characterizedphotoreduction of the oxidized FAD in E. coli photolyasewithout the chromophore MTHF and revealed multiple electron tunnelingpathways to FAD*, especially with the conserved tryptophan triad (W382,W359, and W306 in Figure 1), and determinedall electron-transfer dynamics and time scales.24 For example, in Figure 5C of ref (24), we showed the absorptiontransient probed at 580 nm that was decomposed into five componentsfrom eight species with the reactant (FAD*), intermediates (W382+, W384+, adenine+, W316+,W359+ and W359•.) and final product (W306+). Thus, we completely followed the photoreduction dynamicsof FAD24 to simulate the signals from thedirectly excited FAD* and the decay dynamics of transferred FAD* inFigure 5. (See the SI.) We only need to fit the rise formation component of the transferredintermediate FAD* in 20 ps and all other dynamics of FAD* are completelythe same as the dynamics of FAD photoreduction previously reported.24


Direct determination of resonance energy transfer in photolyase: structural alignment for the functional state.

Tan C, Guo L, Ai Y, Li J, Wang L, Sancar A, Luo Y, Zhong D - J Phys Chem A (2014)

Femtosecond-resolved transient-absorption dynamics probedfrom500 to 700 nm upon excitation at 400 nm. The transients can be systematicallydeconvoluted to three contributions (insets A–D) of the excitedMTHF (dashed blue), the component resulting from the excited FAD fromRET of MTHF* (dashed dark goldenrod), and the signal from the directlyexcited FAD (dashed pink). Note the distinct rise signals reflectingthe intermediate formation through RET of MTHF* and the signal ofthe excited FAD containing its photoreduction dynamics; see the text.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234433&req=5

fig5: Femtosecond-resolved transient-absorption dynamics probedfrom500 to 700 nm upon excitation at 400 nm. The transients can be systematicallydeconvoluted to three contributions (insets A–D) of the excitedMTHF (dashed blue), the component resulting from the excited FAD fromRET of MTHF* (dashed dark goldenrod), and the signal from the directlyexcited FAD (dashed pink). Note the distinct rise signals reflectingthe intermediate formation through RET of MTHF* and the signal ofthe excited FAD containing its photoreduction dynamics; see the text.
Mentions: Figure 5 shows the absorption transients probedfrom 500to 700 nm. Similarly, we observed a distinct rise in tens of picosecondsfrom 580 to 640 nm (also see Figure 5B,C),indicating an intermediate (FAD*) formation. Similar to the analysesabove for the FADH¯ state, all signals are well fit by threeparts: The first one represents the decay of MTHF* absorption or stimulatedemission (negative formation signals in Figure 5B,C) with a time constant of 20 ps determined from the fluorescencequenching dynamics, the second signal results from the directly excitedFAD* species at 400 nm, and the third part is related to the transferredFAD* from MTHF* with a distinct rise and decay pattern. It shouldbe noted that the signal even from the directly excited FAD* at 400nm in Figure 5 is complex and contains manycomponents of different species. We have systematically characterizedphotoreduction of the oxidized FAD in E. coli photolyasewithout the chromophore MTHF and revealed multiple electron tunnelingpathways to FAD*, especially with the conserved tryptophan triad (W382,W359, and W306 in Figure 1), and determinedall electron-transfer dynamics and time scales.24 For example, in Figure 5C of ref (24), we showed the absorptiontransient probed at 580 nm that was decomposed into five componentsfrom eight species with the reactant (FAD*), intermediates (W382+, W384+, adenine+, W316+,W359+ and W359•.) and final product (W306+). Thus, we completely followed the photoreduction dynamicsof FAD24 to simulate the signals from thedirectly excited FAD* and the decay dynamics of transferred FAD* inFigure 5. (See the SI.) We only need to fit the rise formation component of the transferredintermediate FAD* in 20 ps and all other dynamics of FAD* are completelythe same as the dynamics of FAD photoreduction previously reported.24

Bottom Line: Photoantenna is essential to energy transduction in photoinduced biological machinery.A photoenzyme, photolyase, has a light-harvesting pigment of methenyltetrahydrofolate (MTHF) that transfers its excitation energy to the catalytic flavin cofactor FADH¯ to enhance DNA-repair efficiency.We observed 170 ps for excitation energy transferring to the fully reduced hydroquinone FADH¯, 20 ps to the fully oxidized FAD, and 18 ps to the neutral semiquinone FADH(•), and the corresponding orientation factors (κ(2)) were determined to be 2.84, 1.53 and 1.26, respectively, perfectly matching with our calculated theoretical values.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Department of Chemistry and Biochemistry, and Programs of Biophysics, Chemical Physics, and Biochemistry, The Ohio State University , 191 West Woodruff Avenue, Columbus, Ohio 43210, United States.

ABSTRACT
Photoantenna is essential to energy transduction in photoinduced biological machinery. A photoenzyme, photolyase, has a light-harvesting pigment of methenyltetrahydrofolate (MTHF) that transfers its excitation energy to the catalytic flavin cofactor FADH¯ to enhance DNA-repair efficiency. Here we report our systematic characterization and direct determination of the ultrafast dynamics of resonance energy transfer from excited MTHF to three flavin redox states in E. coli photolyase by capturing the intermediates formed through the energy transfer and thus excluding the electron-transfer quenching pathway. We observed 170 ps for excitation energy transferring to the fully reduced hydroquinone FADH¯, 20 ps to the fully oxidized FAD, and 18 ps to the neutral semiquinone FADH(•), and the corresponding orientation factors (κ(2)) were determined to be 2.84, 1.53 and 1.26, respectively, perfectly matching with our calculated theoretical values. Thus, under physiological conditions and over the course of evolution, photolyase has adopted the optimized orientation of its photopigment to efficiently convert solar energy for repair of damaged DNA.

Show MeSH
Related in: MedlinePlus