Replacement of non-heme Fe(II) with Cu(II) in the alpha-ketoglutarate dependent DNA repair enzyme AlkB: spectroscopic characterization of the active site |
| |
Authors: | Bleijlevens Boris Shivarattan Tara Sedgwick Barbara Rigby Stephen E J Matthews Steve J |
| |
Institution: | Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, South Kensington Campus, Biochemistry Building, London SW7 2AZ, UK. |
| |
Abstract: | The bacterial DNA repair enzyme AlkB is an alpha-ketoglutarate (alphaKG) 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-alphaKG 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-alphaKG 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. |
| |
Keywords: | AlkB Non-heme iron Dioxygenase Copper EPR |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|