期刊
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
卷 70, 期 -, 页码 772-779出版社
WILEY-BLACKWELL
DOI: 10.1107/S1399004713033051
关键词
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资金
- Ministry of Education, Science, Sports and Culture of Japan [23580131]
- fund for Kagawa University Young Scientists
- Grants-in-Aid for Scientific Research [24651231, 24119515, 23380197, 23580131, 24657077] Funding Source: KAKEN
Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO from Escherichia coli and laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X- ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O-2 to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O-2-reduction process are discussed.
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