Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli

Sue A. Roberts, Andrzej Weichsel, Gregor Grass, Keshari Thakali, James T. Hazzard, Gordon Tollin, Christopher Rensing, William R. Montfort

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249 Scopus citations

Abstract

CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli. The crystal structure of CueO has been determined to 1.4-Å resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention. This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center. The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site. The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle = 170°) and the third (type II) copper lies only 3.1 Å from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 μM Cu(II). Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k = 4 × 107 M-1·s-1), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 × 107 M-1·s-1, followed by a slow intramolecular electron transfer to T1 copper (k ∼ 10 s-1). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.

Original languageEnglish (US)
Pages (from-to)2766-2771
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number5
DOIs
StatePublished - Mar 5 2002

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