Origin of the transient electron paramagnetic resonance signals in DNA photolyase

Yvonne Gindt, Esther Vollenbroek, Kristi Westphal, Heather Sackett, Aziz Sancar, Gerald T. Babcock

Research output: Contribution to journalArticle

77 Citations (Scopus)

Abstract

DNA photolyase repairs pyrimidine dimer lesions in DNA through light- induced electron donation to the dimer. During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron- oxidized to a semiquinone radical. In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue. This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR. By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin. The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan. We also report a new procedure for the rapid isolation of DNA photolyase.

Original languageEnglish
Pages (from-to)3857-3866
Number of pages10
JournalBiochemistry
Volume38
Issue number13
DOIs
StatePublished - 30 Mar 1999

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Deoxyribodipyrimidine Photo-Lyase
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Electrons
Tryptophan
Pyrimidine Dimers
Photochemistry
Light
Coenzymes
Reducing Agents
Magnetic Fields
DNA Repair
Photochemical reactions
Oxidation-Reduction
Dimers
4,6-dinitro-o-cresol
Catalyst activity
Repair
Magnetic fields
DNA

Cite this

Gindt, Y., Vollenbroek, E., Westphal, K., Sackett, H., Sancar, A., & Babcock, G. T. (1999). Origin of the transient electron paramagnetic resonance signals in DNA photolyase. Biochemistry, 38(13), 3857-3866. https://doi.org/10.1021/bi981191+
Gindt, Yvonne ; Vollenbroek, Esther ; Westphal, Kristi ; Sackett, Heather ; Sancar, Aziz ; Babcock, Gerald T. / Origin of the transient electron paramagnetic resonance signals in DNA photolyase. In: Biochemistry. 1999 ; Vol. 38, No. 13. pp. 3857-3866.
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Gindt, Y, Vollenbroek, E, Westphal, K, Sackett, H, Sancar, A & Babcock, GT 1999, 'Origin of the transient electron paramagnetic resonance signals in DNA photolyase', Biochemistry, vol. 38, no. 13, pp. 3857-3866. https://doi.org/10.1021/bi981191+

Origin of the transient electron paramagnetic resonance signals in DNA photolyase. / Gindt, Yvonne; Vollenbroek, Esther; Westphal, Kristi; Sackett, Heather; Sancar, Aziz; Babcock, Gerald T.

In: Biochemistry, Vol. 38, No. 13, 30.03.1999, p. 3857-3866.

Research output: Contribution to journalArticle

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AB - DNA photolyase repairs pyrimidine dimer lesions in DNA through light- induced electron donation to the dimer. During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron- oxidized to a semiquinone radical. In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue. This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR. By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin. The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan. We also report a new procedure for the rapid isolation of DNA photolyase.

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Gindt Y, Vollenbroek E, Westphal K, Sackett H, Sancar A, Babcock GT. Origin of the transient electron paramagnetic resonance signals in DNA photolyase. Biochemistry. 1999 Mar 30;38(13):3857-3866. https://doi.org/10.1021/bi981191+