Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants

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Abstract
Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyl-tetahydrofolate (5,10-CH+-H4folate). Both chromophores are fluorescent, and either can function as a sensitizer in catalysis. At 77 K separate fluorescence emission bands are observed for FADH2 (.lambda.max = 505 nm, shoulder at 540 nm) and 5,10-CH+-H4 folate (.lambda.max = 465, 440 nm) whereas at 5.degree. C only a shoulder at 505 nm is attributable to FADH2. Formation of an enzyme-substrate complex with various dimer-containing oligothymidylates [UV-oligo(dT)n] quenches the fluorescence due to FADH2 at 5.degree. C or 77 K and also stabilizes FADH2 against air oxidation. The fluorescence of 5,10-CH+-H4folate is unaffected by substrate. Reduction of the pterin chromophore eliminates the chromophore''s fluorescence but does not affect catalytic activity or the ability of substrate to quench FADH2 fluorescence. Quenching of FADH2 fluorescence is fully reversible upon dimer repair. The results are consistent with the proposal that the singlet state of FADH2 functions as an intermediate in catalysis. Fluorometric titrations indicate that the enzyme has a similar affinity for dimers in UV-olgio(dT)4 (KD = 2.5 .times. 10-7 M, .DELTA.G = 8.4 kcal/mol at 5.degree. C) or UV-oligo(dT)6, except for dimers located at the unphosphorylated 3'' end of the oligomers where binding is consiserably weaker. The binding affinity observed with higher homologues [oligo(dT)n, n = 9, 18] (KD .gtoreq. 10-8 M) is within the range previously reported for the binding of photolyase to dimers in DNA (KD .apprx. 10-8 M, .DELTA.G = 10 kcal/mol). Studies with UV-oligo(dT)2 provide an estimate of enzyme binding affinity for the unit pTpT (KD = 5.5 .times. 10-5 M, .DELTA.G = 5.4 kcal/mol) and indicate that about 50% of the binding energy observed with DNA as substrate can be attributed to the binding of the enzyme to this unit. A 1:1 binding stoichiometry with respect to dimer was observed except when the substrate tested contained more than 1 dimer per molecule. Binding affinity was then influenced by the ability of a substrate molecule to accommodate more than 1 molecule of photolyase. The latter was possible with UV-oligo(dT)18 but not with UV-oligo(dT)9.