期刊
JOURNAL OF INORGANIC BIOCHEMISTRY
卷 101, 期 7, 页码 1043-1048出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jinorgbio.2007.03.018
关键词
AlkB; non-heme iron; dioxygenase; copper; EPR
资金
- Cancer Research UK [A5173] Funding Source: Medline
- Wellcome Trust [085464] Funding Source: Medline
The bacterial DNA repair enzyme AlkB is an alpha-ketoglutarate (alpha KG) 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-alpha KG 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-alpha KG 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. (c) 2007 Elsevier Inc. All rights reserved.
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