AFFINITY PROBING OF FLAVIN BINDING-SITES .2. IDENTIFICATION OF A REACTIVE CYSTEINE IN THE FLAVIN DOMAIN OF ESCHERICHIA-COLI DNA PHOTOLYASE

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) i n the flavin domain of Escherichia coli DNA photolyase to form an 8-(c ysteinyl)FAD derivative covalently bound to the protein. About 80% pro tection against covalent attachment with 8-(methylsulfonyl) FAD was ob served in the presence of an equimolar amount of FAD. Flavinylated pho tolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydr ofolate. Comparison of the properties of flavinylated enzyme with phot olyase containing noncovalently bound 8-(methylthio)FAD indicate that a perturbation is necessary to accommodate covalent bond formation. 8- (Methylthio)FAD-reconstituted enzyme exhibits 95% of native activity. The aerobic stability of fully reduced and radical forms of 8-(methylt hio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced en zyme is more easily oxidized by oxygen. The flavin in 8-(methylthio)FA D enzyme or flavinylated photolyase is shielded from solvent. However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in vario us solvents. Enzyme containing noncovalently bound 8-(methylsulfonyl)F AD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment. Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD. The results show that 8-(cysteinyl)FA D in flavinylated photolyase is at or near the normal flavin binding s ite. Although Cys293 in the native structure is probably not in the op timal orientation for nucleophilic attack at C(8), the adjustment need ed for covalent bond formation is not sufficient to grossly interfere with the enzyme's ability to repair DNA or to interact with its antenn a chromophore.