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 language | English (US) |
|---|---|
| Pages (from-to) | 2766-2771 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 99 |
| Issue number | 5 |
| DOIs | |
| State | Published - Mar 5 2002 |
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ASJC Scopus subject areas
- Genetics
- General
Cite this
Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. / Roberts, Sue A; Weichsel, Andrzej; Grass, Gregor; Thakali, Keshari; Hazzard, James T.; Tollin, Gordon; Rensing, Christopher; Montfort, William "Bill".
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 5, 05.03.2002, p. 2766-2771.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli
AU - Roberts, Sue A
AU - Weichsel, Andrzej
AU - Grass, Gregor
AU - Thakali, Keshari
AU - Hazzard, James T.
AU - Tollin, Gordon
AU - Rensing, Christopher
AU - Montfort, William "Bill"
PY - 2002/3/5
Y1 - 2002/3/5
N2 - 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.
AB - 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.
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U2 - 10.1073/pnas.052710499
DO - 10.1073/pnas.052710499
M3 - Article
C2 - 11867755
AN - SCOPUS:0037022682
VL - 99
SP - 2766
EP - 2771
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 5
ER -