Resonance Raman and UV-Vis spectroscopic characterization of FADH in the complex of photolyase with UV-damaged DNA

Johannes Schelvis, Meghan Ramsey, Olga Sokolova, Celia Tavares, Christine Cecala, Katelyn Connell, Stacey Wagner, Yvonne Gindt

Research output: Contribution to journalArticleResearchpeer-review

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Abstract

Escherichia coli photolyase uses blue light to repair cyclobutane pyrimidine dimers which are formed upon irradiation of DNA with ultraviolet (UV) light. E. coli photolyase is a flavoenzyme which contains a flavin adenine dinucleotide (FAD) in its active site and a 5,10-methenyltetrahydrofolate (MTHF) as a light-harvesting pigment. In the isolated enzyme, the FAD cofactor is present as a stable neutral radical semiquinone (FADH). In this paper, we investigate the interaction between photolyase and UV-damage DNA by using resonance Raman and UV-vis spectroscopy. Substrate binding results in intensity changes and frequency shifts of the FADH vibrations and also induces electrochromic shifts of the FADH electronic transitions because of the substrate electric dipole moment. The intensity changes in the resonance Raman spectra can be largely explained by changes in the Raman excitation profiles because of the electrochromic shift. The size of the electrochromic shift suggests that the substrate binding geometry is similar to that of oxidized FAD in reconstituted photolyase. The frequency changes are partially a manifestation of the vibrational Stark effect induced by the substrate electric dipole moment but also because of small perturbations of the hydrogen-bonding environment of FADH upon substrate binding. Furthermore, differences in the resonance Raman spectra of MTHF-containing photolyase and of an MTHF-less mutant suggests that MTHF may play a structural role in stabilizing the active site of photolyase while comparison to other flavoproteins indicates that the FAD cofactor has a strong hydrogen-bonding protein environment. Finally, we show that the electrochromic shift can be used as a direct method to measure photolyase-substrate binding kinetics.

Original languageEnglish
Pages (from-to)12352-12362
Number of pages11
JournalJournal of Physical Chemistry B
Volume107
Issue number44
StatePublished - 6 Nov 2003

Fingerprint

Deoxyribodipyrimidine Photo-Lyase
DNA
deoxyribonucleic acid
adenines
Flavin-Adenine Dinucleotide
Substrates
Electric dipole moments
shift
electric moments
Escherichia coli
electric dipoles
Raman scattering
Hydrogen bonds
dipole moments
Raman spectra
Stark effect
cyclobutane
Pyrimidine Dimers
Flavoproteins
ultraviolet spectroscopy

Cite this

Schelvis, Johannes ; Ramsey, Meghan ; Sokolova, Olga ; Tavares, Celia ; Cecala, Christine ; Connell, Katelyn ; Wagner, Stacey ; Gindt, Yvonne. / Resonance Raman and UV-Vis spectroscopic characterization of FADH in the complex of photolyase with UV-damaged DNA. In: Journal of Physical Chemistry B. 2003 ; Vol. 107, No. 44. pp. 12352-12362.
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title = "Resonance Raman and UV-Vis spectroscopic characterization of FADH• in the complex of photolyase with UV-damaged DNA",
abstract = "Escherichia coli photolyase uses blue light to repair cyclobutane pyrimidine dimers which are formed upon irradiation of DNA with ultraviolet (UV) light. E. coli photolyase is a flavoenzyme which contains a flavin adenine dinucleotide (FAD) in its active site and a 5,10-methenyltetrahydrofolate (MTHF) as a light-harvesting pigment. In the isolated enzyme, the FAD cofactor is present as a stable neutral radical semiquinone (FADH•). In this paper, we investigate the interaction between photolyase and UV-damage DNA by using resonance Raman and UV-vis spectroscopy. Substrate binding results in intensity changes and frequency shifts of the FADH• vibrations and also induces electrochromic shifts of the FADH• electronic transitions because of the substrate electric dipole moment. The intensity changes in the resonance Raman spectra can be largely explained by changes in the Raman excitation profiles because of the electrochromic shift. The size of the electrochromic shift suggests that the substrate binding geometry is similar to that of oxidized FAD in reconstituted photolyase. The frequency changes are partially a manifestation of the vibrational Stark effect induced by the substrate electric dipole moment but also because of small perturbations of the hydrogen-bonding environment of FADH• upon substrate binding. Furthermore, differences in the resonance Raman spectra of MTHF-containing photolyase and of an MTHF-less mutant suggests that MTHF may play a structural role in stabilizing the active site of photolyase while comparison to other flavoproteins indicates that the FAD cofactor has a strong hydrogen-bonding protein environment. Finally, we show that the electrochromic shift can be used as a direct method to measure photolyase-substrate binding kinetics.",
author = "Johannes Schelvis and Meghan Ramsey and Olga Sokolova and Celia Tavares and Christine Cecala and Katelyn Connell and Stacey Wagner and Yvonne Gindt",
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Schelvis, J, Ramsey, M, Sokolova, O, Tavares, C, Cecala, C, Connell, K, Wagner, S & Gindt, Y 2003, 'Resonance Raman and UV-Vis spectroscopic characterization of FADH in the complex of photolyase with UV-damaged DNA', Journal of Physical Chemistry B, vol. 107, no. 44, pp. 12352-12362.

Resonance Raman and UV-Vis spectroscopic characterization of FADH in the complex of photolyase with UV-damaged DNA. / Schelvis, Johannes; Ramsey, Meghan; Sokolova, Olga; Tavares, Celia; Cecala, Christine; Connell, Katelyn; Wagner, Stacey; Gindt, Yvonne.

In: Journal of Physical Chemistry B, Vol. 107, No. 44, 06.11.2003, p. 12352-12362.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Resonance Raman and UV-Vis spectroscopic characterization of FADH• in the complex of photolyase with UV-damaged DNA

AU - Schelvis, Johannes

AU - Ramsey, Meghan

AU - Sokolova, Olga

AU - Tavares, Celia

AU - Cecala, Christine

AU - Connell, Katelyn

AU - Wagner, Stacey

AU - Gindt, Yvonne

PY - 2003/11/6

Y1 - 2003/11/6

N2 - Escherichia coli photolyase uses blue light to repair cyclobutane pyrimidine dimers which are formed upon irradiation of DNA with ultraviolet (UV) light. E. coli photolyase is a flavoenzyme which contains a flavin adenine dinucleotide (FAD) in its active site and a 5,10-methenyltetrahydrofolate (MTHF) as a light-harvesting pigment. In the isolated enzyme, the FAD cofactor is present as a stable neutral radical semiquinone (FADH•). In this paper, we investigate the interaction between photolyase and UV-damage DNA by using resonance Raman and UV-vis spectroscopy. Substrate binding results in intensity changes and frequency shifts of the FADH• vibrations and also induces electrochromic shifts of the FADH• electronic transitions because of the substrate electric dipole moment. The intensity changes in the resonance Raman spectra can be largely explained by changes in the Raman excitation profiles because of the electrochromic shift. The size of the electrochromic shift suggests that the substrate binding geometry is similar to that of oxidized FAD in reconstituted photolyase. The frequency changes are partially a manifestation of the vibrational Stark effect induced by the substrate electric dipole moment but also because of small perturbations of the hydrogen-bonding environment of FADH• upon substrate binding. Furthermore, differences in the resonance Raman spectra of MTHF-containing photolyase and of an MTHF-less mutant suggests that MTHF may play a structural role in stabilizing the active site of photolyase while comparison to other flavoproteins indicates that the FAD cofactor has a strong hydrogen-bonding protein environment. Finally, we show that the electrochromic shift can be used as a direct method to measure photolyase-substrate binding kinetics.

AB - Escherichia coli photolyase uses blue light to repair cyclobutane pyrimidine dimers which are formed upon irradiation of DNA with ultraviolet (UV) light. E. coli photolyase is a flavoenzyme which contains a flavin adenine dinucleotide (FAD) in its active site and a 5,10-methenyltetrahydrofolate (MTHF) as a light-harvesting pigment. In the isolated enzyme, the FAD cofactor is present as a stable neutral radical semiquinone (FADH•). In this paper, we investigate the interaction between photolyase and UV-damage DNA by using resonance Raman and UV-vis spectroscopy. Substrate binding results in intensity changes and frequency shifts of the FADH• vibrations and also induces electrochromic shifts of the FADH• electronic transitions because of the substrate electric dipole moment. The intensity changes in the resonance Raman spectra can be largely explained by changes in the Raman excitation profiles because of the electrochromic shift. The size of the electrochromic shift suggests that the substrate binding geometry is similar to that of oxidized FAD in reconstituted photolyase. The frequency changes are partially a manifestation of the vibrational Stark effect induced by the substrate electric dipole moment but also because of small perturbations of the hydrogen-bonding environment of FADH• upon substrate binding. Furthermore, differences in the resonance Raman spectra of MTHF-containing photolyase and of an MTHF-less mutant suggests that MTHF may play a structural role in stabilizing the active site of photolyase while comparison to other flavoproteins indicates that the FAD cofactor has a strong hydrogen-bonding protein environment. Finally, we show that the electrochromic shift can be used as a direct method to measure photolyase-substrate binding kinetics.

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