Electronic and Functional Structure of Copper in Plant Cu/Zn Superoxide Dismutase with Combined Site-directed Mutagenesis and Electron Paramagnetic Resonance |
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Authors: | Zuo-Jun LIU Ming CHEN Sheng-Wei HUANG Ming-Hao LI Jin-Yan HOU Ying-Ji MAO Wei TONG Ji-Hu SU Li-Fang WU |
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Institution: | 1. Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. CAS Key Laboratory of Microscale Magnetic Resonance, Department of Modern Physics, University of Science and Technology of China, Hefei 230026, China;4. Agricultural Engineering Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230001, China |
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Abstract: | Arabidopsis thaliana copper-zinc superoxide dismutase 1 (AtSOD1) is a typical metalloenzyme conferring cellular protection against the excessive accumulation of toxic reactive oxygen species, and is therefore considered as a critical protein. However, the structure and function of the vital amino acids around the active site of AtSOD1 remain poorly understood. Herein, the coordinated geometry of the catalytic center in AtSOD1 was reconstructed by electron paramagnetic resonance (EPR) technique, and it was found to be composed of copper and four histidine (H) residues using site-directed mutagenesis. Analysis of the mutants showed that H45 and H62 play essential roles in the catalytic reaction, and H119 plays an accessary role in facilitating substrate or proton transfer. The results indicated that the redox change of the Cu ion and the overall enzymatic activity of the protein were sustained by the H45-Cu-H62 core structure. In contrast, the residue H47 showed nearly no effect on the SOD catalytic activity. These data should contribute to a deeper understanding of the catalytic mechanism of the enzyme, and provide a new approach for the effective molecular modification of copper/zinc SODs to facilitate further research in this field. |
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Keywords: | Metalloenzyme Copper-zinc superoxide dismutase 1 Electron paramagnetic resonance Site-directed mutagenesis Reactive oxygen species |
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