Abstract:
:The folate chromophore in native Escherichia coli DNA photolyase ([6R]-5,10-CH+-H4Pte(Glu)n=3-6) serves as an antenna, transferring light energy to the fully reduced flavin (FADH2) reaction center at high efficiency (EET = 0.92). Apophotolyase reconstituted after an overnight incubation with [6R,S]-5,10-CH+-H4folate (a monoglutamate analogue of the native cofactor) contains equimolar amounts of the [6R]- and [6S]-isomers, suggesting similar binding affinities. A rapid, biphasic increase in fluorescence (approximately 100-fold) is observed upon binding of 5,10-CH+-H4folate to apophotolyase at 5 degrees C; the [6S]-isomer binds about 25-fold faster than the [6R]-isomer. Although identical absorption and fluorescence emission maxima are observed for enzyme reconstituted with [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate, folate fluorescence quantum yield values vary depending on the stereochemical configuration at the 6 position (theta = 0.18, 0.82, or 0.46, respectively, at 5 degrees C), a feature not seen with free folate. The fluorescence of enzyme-bound folate is quenched upon flavin binding; the efficiency of quenching by flavin radical (EQ = 0.96) or FADH2 (EQ = 0.89) is the same for both folate isomers. In contrast, energy transfer from folate to FADH2 is sensitive to the stereochemical configuration at the 6 position. The efficiency of energy transfer observed for enzyme containing FADH2 and [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate (theta = 0.26, 0.66, or 0.44, respectively) is directly proportional to the fluorescence quantum yield observed for folate in the absence of FADH2, as expected for Förster-type energy transfer. Although less efficient, the unnatural [6S]-isomer is catalytically functional, a feature not previously observed with other folate-dependent enzymes. Fluorescence quantum yield studies at 77 K with free (theta = 0.67) and enzyme-bound (theta = 1.0) folate suggest that differences in solvent exposure may contribute to the fluorescence efficiency differences observed with the enzyme-bound folate isomers at 5 degrees C.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Lipman RS,Jorns MSdoi
10.1021/bi9605548subject
Has Abstractpub_date
1996-06-18 00:00:00pages
7968-73issue
24eissn
0006-2960issn
1520-4995pii
bi9605548journal_volume
35pub_type
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