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‡, and William R. Montfort*§ Departments of *Biochemistry and Molecular Biophysics, and ‡Soil, Water, and Environmental Science, University of Arizona, Tucson, AZ 85721 Communicated by John H. Law, University of Arizona, Tucson, AZ, December 31, 2001 (received for review December 4, 2001) CueO (YacK), a multicopper oxidase, is part of the copper-regula- diseases (12). The Wilson disease ATPase transports copper into tory cue operon in Escherichia coli. The crystal structure of CueO the hepatocyte secretory pathway for incorporation into ceru- has been determined to 1.4-Å resolution by using multiple anom- loplasmin. Thus, the cue pathway in bacteria may be functionally alous dispersion phasing and an automated building procedure similar to both copper and iron homeostasis activities in humans. that yielded a nearly complete model without manual interven- MCOs couple four one-electron substrate oxidation steps to tion. This is the highest resolution multicopper oxidase structure the four-electron reduction of dioxygen to water (2). This yet determined and provides a particularly clear view of the four reaction has been best studied in the proteins laccase (E.C. coppers at the catalytic center. The overall structure is similar to 1.10.3.2), AO (E.C.1.10.3.3), and ceruloplasmin (E.C. 1.16.3.1). those of laccase and ascorbate oxidase, but contains an extra MCOs typically contain four copper atoms, one type I (T1) or 42-residue insert in domain 3 that includes 14 methionines, nine of ‘‘blue’’ copper site, and three other atoms in a trinuclear cluster which lie in a helix that covers the entrance to the type I (T1, blue) consisting of one type II (T2) or ‘‘normal’’ copper, and two type copper site. The trinuclear copper cluster has a conformation not III (T3) or ‘‘binuclear’’ coppers (2). Molecular oxygen binds at previously seen: the Cu-O-Cu binuclear species is nearly linear the trinuclear site and is reduced to water through four single- ؍ (Cu-O-Cu bond angle 170°) and the third (type II) copper lies only electron transfer steps from the T1 site, located about 13 Å away. 3.1 Å from the bridging oxygen. CueO activity was maximal at pH Some MCOs, such as laccase, have little specificity and can ␮ 6.5 and in the presence of >100 M Cu(II). Measurements of oxidize a large variety of substrates, whereas others, such as AO, intermolecular and intramolecular electron transfer with laser flash are more specific. photolysis in the absence of Cu(II) show that, in addition to the The crystal structures of three enzymes in this family, laccase normal reduction of the T1 copper, which occurs with a slow rate (13), AO (14), ceruloplasmin (15), and a fourth enzyme con- ؋ 107 M؊1⅐s؊1), a second electron transfer process occurs to 4 ؍ k) taining a T1 copper and a second copper center, nitrite reduc- ؋ 107 9 ؍ an unknown site, possibly the trinuclear cluster, with k tase (16), have been determined at resolutions ranging from 1.9 M؊1⅐s؊1, followed by a slow intramolecular electron transfer to T1 to 3.1 Å. In laccase and AO, each polypeptide chain adopts a copper (k ϳ10 s؊1). These results suggest the methionine-rich helix three-domain structure with the trinuclear copper center located blocks access to the T1 site in the absence of excess copper. in the interior of the protein between domains I and III. The T1 copper center is in domain III and somewhat buried. There is a ulticopper oxidases (MCOs) are prominent in all king- shallow depression at the protein surface where substrates can Mdoms and play a critical role in iron metabolism and approach the T1 copper site and where ligand discrimination copper homeostasis. In mammals, ceruloplasmin functions as a occurs in AO. The T1 copper center and the trinuclear copper ferroxidase, catalyzing the conversion of Fe(II) to Fe(III), and center are connected by the amino acid sequence HCH, where is the major copper-containing protein in plasma (reviewed in the two histidine residues ligate the two T3 copper atoms of the refs. 1 and 2). Animals deficient in ceruloplasmin are anemic, trinuclear cluster and the cysteine is a ligand of the T1 copper. presumably because of the loss of ferroxidase activity. Copper is CueO (formerly YacK), identified as a 53.4-kDa homologue of both essential and toxic, because of metal-catalyzed protein laccase (17), has been shown to oxidize a wide variety of oxidation, and improper copper metabolism in humans is linked substrates including 2,6-dimethyoxyphenol (DMP), enterobactin to several diseases, including Menkes and Wilson diseases (3), (a catecholate siderophore produced by E. coli), and ferrous iron Alzheimer’s disease, and other aging disorders (4). In yeast, the (10, 11), and depends on oxygen for activity. Intriguingly, very MCO Fet3p has ferroxidase activity and is required for iron little oxidase activity occurs in the absence of excess copper. The uptake through generation of ferric iron, the form thought to be mechanism by which CueO protects against copper stress in cells used by the yeast iron transporter Ftr1p (5, 6). In plants, is not fully understood, but is unlikely to be solely because of ascorbate oxidase (AO) and laccases (also a fungal enzyme) copper binding (8, 9). oxidize various phenolic substrates that, in the case of laccase, Ϸ lead to protective polymers (7). An unusual feature of the CueO sequence is a region of 45 In bacteria, the genes and proteins necessary for copper aa at the beginning of domain III (355–400) that includes 14 homeostasis are just now coming to light. Studies of copper methionines and could provide binding sites for exogenous toxicity in Escherichia coli have unveiled two regulatory path- ways, the cus system (for Cu-sensing) and cue system (for Abbreviations: MCO, multicopper oxidase; AO, ascorbate oxidase; T1, type I; T2, type II; T3, Cu-efflux) (8). The cus operon codes for three proteins that form type III; DMP, dimethyoxyphenol; 5-dRFH⅐, 5-deazariboflavin. a multiunit transport complex that spans the periplasmic space Data deposition: The atomic coordinates and structure factors have been deposited in the and links the inner and outer membranes. The complex is Protein Data Bank, www.rcsb.org (PDB ID code 1KV7). proposed to function in copper efflux (8, 9) and is most active †S.A.R. and A.W. contributed equally to this work. under anaerobic growth conditions (8). The cue operon codes for §To whom reprint requests should be addressed. E-mail: [email protected]. two proteins, CueO, a MCO residing in the periplasmic space (8, The publication costs of this article were defrayed in part by page charge payment. This 10, 11), and CopA, a copper efflux P-type ATPase related to the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. proteins that, when dysfunctional, cause Menkes and Wilson §1734 solely to indicate this fact. 2766–2771 ͉ PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.052710499 Downloaded by guest on September 29, 2021 Table 1. X-ray data measurement and phase determination Native Peak Remote Inflection Data measurement Wavelength (Å) 1.0000 1.3772 1.1922 1.3804 Resolution (Å)26–1.40 26–2.14 26–2.14 26–2.14 Observed reflections 733,346 91,035 89,158 91,943 Unique reflections 88,826 24,997 24,462 25,191 Completeness (%)* 98.5͞87.1 97.2͞99.0 96.2͞99.6 97.6͞98.9 Rsym* 0.064͞0.280 0.024͞0.039 0.024͞0.041 0.025͞0.044 I͞␴(I)* 7.0͞2.3 19.0͞13.6 16.5͞12.2 18.5͞12.4 Phasing Phasing power† 0.8͞0.53 1.29͞1.00 † Rcullis 0.88͞0.88 0.76͞0.67 Rcullis anomalous 0.76 0.82 *Overall͞outermost shell. †Acentric͞centric. copper ions. A similar methionine-rich region also is seen in the surface. Generation of the neutral semiquinone of 5-deaz- PcoA and CopA (Pseudomonas syringae), two proteins also ariboflavin (5-dRFH⅐) was accomplished by using a ns flash from ϭ implicated in copper tolerance, leading to the suggestion that this aN2-pumped dye laser (emission wavelength 395 nm). Kinetic methionine-rich region is important for copper tolerance in traces were analyzed by using KINFIT (OLIS, Jefferson, GA). bacteria (18). Here, we report the crystal structure of CueO, which shows the methionine-rich region to be largely helical and Crystallization and Data Collection. Monoclinic crystals of CueO to cover the substrate-binding region. We also report both grew as rectangular blue plates by the hanging drop vapor steady-state oxidation and transient intramolecular electron diffusion method from 20% PEG 4000, 200 mM ammonium transfer kinetics. The CueO structure, at 1.4 Å, is the highest acetate, 100 mM sodium acetate, pH 4.6 at room temperature. resolution MCO structure determined for any species. The The crystals belong to space group P21 with unit cell dimensions kinetic studies support a model where excess copper alters access a ϭ 49.8 Å, b ϭ 90.5 Å, c ϭ 53.1 Å, ␤ ϭ 102.9°, and one molecule to the T1 site and suggests that intramolecular electron transfer in the asymmetric unit. Before data collection, a 0.4 ϫ 0.3 ϫ between the trinuclear cluster and the T1 site is about 16-fold 0.05-mm crystal was transferred to 50% PEG 4000 and frozen in slower than in AO.