Replacement of non-heme Fe(II) with Cu(II) in the α-ketoglutarate dependent DNA repair enzyme AlkB: Spectroscopic characterization of the active site

Abstract: The bacterial DNA repair enzyme AlkB is an α-ketoglutarate (αKG) dependent non-heme Fe(II) containing dioxygenase. Here we describe, for the first time, the preparation of a Cu(II)-reconstituted form of AlkB in various complexes. Spectroscopic characterization showed correct AlkB folding upon incorporation of Cu(II) in the active site. The Cu site was classified as a type 2 site by EPR spectroscopy. The accessibility of the active site metal was studied using imidazole as a probe. Although addition of imidazole did not change the EPR spectrum of the AlkB–Cu–αKG complex, the spectrum of the AlkB–Cu–succinate complex clearly changed, indicating binding of imidazole at the Cu site. Binding of substrate (methylated DNA) to the AlkB–Cu–αKG complex did not induce changes in the EPR spectrum, demonstrating that the substrate does not bind in the immediate vicinity of the metal centre. This work provides a basis for advanced EPR approaches aimed at studying the interactions and dynamics of AlkB complexes in solution. [Copyright &y& Elsevier]