Light‐induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy

Cyclobutane‐type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme‐catalysed reaction is believed to be initiated by the light‐induced transfer of an electron from the anionic FADH− chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue‐coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light‐driven DNA repair reaction and can be reduced to the enzymatically active FADH− form by red‐light irradiation. Difference Fourier transform infrared (FT‐IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH− form of the enzyme. Preliminary band assignments are based on experiments with 15N‐labelled enzyme and on experiments with D2O as solvent. Difference FT‐IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light‐driven photolyase catalysis. This study provides the basis for future time‐resolved FT‐IR studies which are aimed at an elucidation of a detailed molecular picture of the light‐driven DNA repair process.