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

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-A 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 degrees) and the third (type II) copper lies only 3.1 A from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 microM 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 x 10(7) M(-1)x (-1)), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 x 10(7) M(-1) x (-1), followed by a slow intramolecular electron transfer to T1 copper (k approximately 10 s(-1)). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.

Figure 1

Transient absorbance changes obtained at 610 nm for anaerobic laser flash reduction of 35 μM CueO by 5-deazariboflavin semiquinone in 100 mM sodium phosphate buffer at pH = 8.0. (A) Kinetic trace for the intramolecular reduction of the T1 Cu. The solid line represents a single exponential fit to the data with a rate constant of 12 s⁻¹. (B) Kinetic trace for the bimolecular (direct) reduction of the T1 Cu by 5-dRFH⋅. The solid line represents a single exponential fit to the data with a k_obs = 180 s⁻¹.

Figure 2

Representative electron density (2 F_o − F_c α_c) after automatic model building and one round of refinement, before manual intervention. The model shown is in the region surrounding the trinuclear copper center and was built automatically with ARP/WARP.

Figure 3

(A) Ribbon drawing of CueO, with the T1 copper and Cu₃O cluster shown as space-filling spheres. The three related domains are colored separately, and the methionine-rich helix is located in the lower right. (B) Ribbon drawing of AO (PDB entry 1AOZ), an MCO with a more accessible T1 site. (C) Close-up of the methionine-rich region.

Figure 4

Stereoview showing the geometry of the T1 and trinuclear copper sites. Shown are the copper atoms (cyan), oxygen atoms (red), HCH residues (499–501), nitrogens (blue), and sulfurs (yellow) from ligating histidines, cysteines, and methionines. T1 Cu is ligated to His-443, His-503, Cys-500, and Met-510. T2 Cu is ligated to His-101, His-446, and a water molecule. Cu2 is ligated to His-103, His-141, His-501, and the bridging oxygen. Cu3 is ligated to His-143, His-448, His-499, and the bridging oxygen.