Reaction mechanisms of the multicopper oxidase CueO from escherichia coli support its functional role as a cuprous oxidase

Djoko, Karrera Y., Chong, Lee Xin, Wedd, Anthony G. and Xiao, Zhiguang (2010) Reaction mechanisms of the multicopper oxidase CueO from escherichia coli support its functional role as a cuprous oxidase. Journal of American Chemical Society, 132 6: 2005-2015. doi:10.1021/ja9091903

Author Djoko, Karrera Y.
Chong, Lee Xin
Wedd, Anthony G.
Xiao, Zhiguang
Title Reaction mechanisms of the multicopper oxidase CueO from escherichia coli support its functional role as a cuprous oxidase
Journal name Journal of American Chemical Society   Check publisher's open access policy
ISSN 0002-7863
Publication date 2010-01-20
Sub-type Article (original research)
DOI 10.1021/ja9091903
Volume 132
Issue 6
Start page 2005
End page 2015
Total pages 11
Place of publication United States
Publisher American Chemical Society
Language eng
Formatted abstract
CueO from Escherichia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions. It features four copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine-rich insert which includes a helix that blocks physical access to the T1 site and which provides an extra labile site T4 adjacent to the T1 center. This T4 site is required for CueO function. Like many MCOs, CueO exhibits phenol oxidase activity with broad substrate specificity. Maximal activity with model substrate 2,6-dimethoxyphenol required stoichiometric occupation of T4 by CuII (notation: Cu II-CueO). This was achieved in Mops buffer which has little affinity for Cu2+. However, pH buffers that bind or precipitate Cu 2+ (Tris, BisTris, and KPi) generated enzyme with a vacant T4 site (notation: □-CueO) which has no phenol oxidase activity. Addition of excess Cu2+ effectively generated a Cu2+ buffer and recovered the activity partially or completely, depending upon the specific pH buffer. This phenomenon allowed reliable estimation of the affinity of T4 for Cu II: KD = 5.5 × 10-9 M. CueO is involved in copper tolerance and has been suggested to be a cuprous oxidase. The anion [CuI(Bca)2]3- (Bca = bicinchoninate) acted as a novel chromophoric substrate. It is a robust reagent, being air-stable and having a CuI affinity comparable to those of periplasmic Cu I binding proteins. The influences of pH buffer composition and of excess Cu2+ on cuprous oxidation were diametrically opposite to those seen for phenol oxidation, suggesting that □-CueO, not Cu II-CueO, is the resting form of the cuprous oxidase. Steady-state kinetics demonstrated that the intact anion [CuI(Bca) 2]3-, not "free" Cu+, is the substrate that donates CuI directly to T4. The data did not follow classical Michaelis-Menten kinetics but could be fitted satisfactorily by an extension that considered the effect of free ligand Bca. The Km term consists of two components, allowing estimation of the transient affinity of T4 for CuI: KD = 1.3 × 10-13 M. It may be concluded that CuI carried by [CuI(Bca)2] 3- is oxidized only upon complete transfer of CuI to T4. The transfer is required to induce a negative shift in the copper reduction potential to allow oxidation and electron transfer to the T1 site. The results provide compelling evidence that CueO is a cuprous oxidase. The new approach will have significant application to the study of metallo-oxidase enzymes.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
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Created: Tue, 20 Sep 2011, 14:22:35 EST by Karrera Djoko on behalf of School of Chemistry & Molecular Biosciences