Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase.
The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+)-H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)
Lipman
R S
RS
Department of Biological Chemistry, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
Jorns
M S
MS
D000693:Anaerobiosis; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D013763:Tetrahydrofolates / Q000378:metabolism
D013763:Tetrahydrofolates; D005182:Flavin-Adenine Dinucleotide; C018092:5,10-methenyltetrahydrofolate; D004255:Deoxyribodipyrimidine Photo-Lyase