title: Replacement of non-heme Fe(II) with Cu(II) in the alpha-ketoglutarate dependent DNA repair enzyme AlkB: spectroscopic characterization of the active site.
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.
authors.affiliation: Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, South Kensington Campus, Biochemistry Building, London SW7 2AZ, UK.
authors.forename: Boris
authors.initials: B
authors.lastname: Bleijlevens
authors.forename: Tara
authors.initials: T
authors.lastname: Shivarattan
authors.forename: Barbara
authors.initials: B
authors.lastname: Sedgwick
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Matthews
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D004260:DNA Repair; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
chemical_list: D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D006418:Heme; D003300:Copper; D007501:Iron; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli