Evidence of powerful substrate electric fields in DNA photolyase: implications for thymidine dimer repair.

DNA photolyase is a flavoprotein that repairs cyclobutylpyrimidine dimers by ultrafast photoinduced electron transfer. One unusual feature of this enzyme is the configuration of the FAD cofactor, where the isoalloxazine and adenine rings are nearly in vdW contact. We have measured the steady-state and transient absorption spectra and excited-state decay kinetics of oxidized (FAD-containing, folate-depleted) Escherichia coli DNA photolyase with and without dinucleotide and polynucleotide single-stranded thymidine dimer substrates. The steady-state absorption spectrum for the enzyme-polynucleotide substrate complex showed a blue shift, as seen previously by Jorns et al. (1). No shift was observed for the dinucleotide substrate, suggesting that there are significant differences in the binding geometry of dinucleotide versus polynucleotide dimer lesions. Evidence was obtained from transient absorption experiments for a long-lived charge-transfer complex involving the isoalloxazine of the FAD cofactor. No evidence of excited-state quenching was measurable upon binding either substrate. To explain these data, we hypothesize the existence of a large substrate electric field in the cavity containing the FAD cofactor. A calculation of the magnitude and direction of this dipolar electric field is consistent with electrochromic band shifts for both S(0) --> S(1) and S(0) --> S(2) transitions. These observations suggest that the substrate dipolar electric field may be a critical component in its electron-transfer-mediated repair by photolyase and that the unique relative orientation of the isoalloxazine and adenine rings may have resulted from the consequences of the dipolar substrate field.