Origin of the transient electron paramagnetic resonance signals in DNA photolyase.
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.
Gindt
Y M
YM
Department of Chemistry, Michigan State University, East Lansing 48824, USA.
Vollenbroek
E
E
Westphal
K
K
Sackett
H
H
Sancar
A
A
Babcock
G T
GT
D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000737:chemistry; D006358:Hot Temperature; D010084:Oxidation-Reduction; D013113:Spin Labels; D014364:Tryptophan / Q000737:chemistry
D013113:Spin Labels; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D014364:Tryptophan; D004255:Deoxyribodipyrimidine Photo-Lyase