Visualization of dioxygen bound to copper during enzyme catalysis
C M Wilmot1, J Hajdu, M J McPherson
1Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Science (New York, N.Y.)
|November 27, 1999
Summary
Copper amine oxidase structures reveal oxygen reduction mechanisms. Hydrolysis regeneration of the quinone cofactor is inhibited by product aldehyde, controlling reaction speed.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Copper-containing quinoprotein amine oxidases are crucial enzymes catalyzing amine oxidation.
- Understanding their reaction mechanisms, particularly the oxidative half, is vital for biochemical insights.
Purpose of the Study:
- To elucidate the structural basis of the oxidative half-reaction in Escherichia coli amine oxidase.
- To investigate the roles of the quinone cofactor and catalytic residues in oxygen reduction and product release.
Main Methods:
- X-ray crystallography was used to determine the structures of three related species at high resolution (2.1–2.4 Å).
- Crystals were prepared under anaerobic and aerobic conditions after substrate exposure and freeze-trapped.
- Single-crystal spectrophotometry was employed to assess the oxidation state of the quinone cofactor.
Main Results:
- The structures identified the binding site of dioxygen and elucidated proton transfer pathways essential for oxygen reduction.
- Regeneration of the quinone cofactor from the iminoquinone intermediate involves hydrolysis mediated by Asp383.
- Product aldehyde was found to inhibit this hydrolysis step, indicating product release as rate-limiting.
Conclusions:
- The study provides detailed structural insights into the oxygen reduction mechanism of copper amine oxidase.
- Asp383 acts as the catalytic base for quinone regeneration via hydrolysis.
- Product inhibition by aldehyde highlights a key regulatory step in the enzyme's catalytic cycle.
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