The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro

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Abstract
Escherichia coli DNA photolyase is a flavoprotein that when purified is blue in color and contains a stable neutral radical FAD (E-FADH.). In the presence of a suitable electron donor (i.e., thiols, tyrosine, or NADH) the radical FAD absorbs visible light and undergoes photoreduction to the fully reduced FAD (E-FADH2). The in vitro quantum yield of dimer repair for E-FADH is 0.07 while that of E-FADH2 approaches the in vivo value of 1. Electron paramagnetic resonance studies on whole cells indicate that the in vivo form of photolyase is E-FADH2 with enzyme containing radical FAD generated predominantly during the ammonium sulfate precipitation step of the purification. Activity measurements of E-FADH. using long-wavelength photoreactivating light indicate that enzyme containing FAD in the radical form is not active in dimer repair. Dimer repair observed with E-FADH. at shorter wavelengths is probably photoreduction of E-FADH. followed by dimer repair by E-FADH2.