Publications

• Sequential reconstitution of copper sites in the multicopper oxidase CueO.

  Galli I., Musci G., Bonaccorsi di Patti M.C.

  CueO belongs to the family of multicopper oxidases which are characterized by the presence of multiple copper-binding sites with different structural and functional properties. These enzymes share the ability to couple the one-electron oxidation of substrate to reduction of oxygen to water by way ... >> More

  CueO belongs to the family of multicopper oxidases which are characterized by the presence of multiple copper-binding sites with different structural and functional properties. These enzymes share the ability to couple the one-electron oxidation of substrate to reduction of oxygen to water by way of a functional unit composed of a mononuclear type 1 blue copper site, which is the entry site for electrons, and of a trinuclear copper cluster formed by type 2 and binuclear type 3 sites, where oxygen binding and reduction take place. The mechanism of copper incorporation in CueO has been investigated by optical and EPR spectroscopy. The results indicate unambiguously that the process is sequential, with type 1 copper being the first to be reconstituted, followed by type 2 and type 3 sites. << Less

  J Biol Inorg Chem 9:90-95(2004) [PubMed] [EuropePMC]

• CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli.

  Grass G., Rensing C.

  The putative multi-copper oxidase CueO had previously been implicated in intrinsic copper resistance in Escherichia coli. In this report we showed that the presence of CueO in the periplasm protected alkaline phosphatase from copper-induced damage. CueO contained four copper atoms per molecule and ... >> More

  The putative multi-copper oxidase CueO had previously been implicated in intrinsic copper resistance in Escherichia coli. In this report we showed that the presence of CueO in the periplasm protected alkaline phosphatase from copper-induced damage. CueO contained four copper atoms per molecule and displayed spectroscopic properties typical of blue copper oxidases. CueO catalyzed the oxidation of p-phenylenediamine (pPD), 2,6-dimethoxyphenol (DMP) and exhibited ferroxidase activity in vitro. << Less

  Biochem. Biophys. Res. Commun. 286:902-908(2001) [PubMed] [EuropePMC]

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

  Roberts S.A., Weichsel A., Grass G., Thakali K., Hazzard J.T., Tollin G., Rensing C., Montfort W.R.

  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 ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 99:2766-2771(2002) [PubMed] [EuropePMC]

• The functional roles of the three copper sites associated with the methionine-rich insert in the multicopper oxidase CueO from E. coli.

  Cortes L., Wedd A.G., Xiao Z.

  CueO from Escherichia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions. It features the four typical copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine- and histidine-rich ... >> More

  CueO from Escherichia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions. It features the four typical copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine- and histidine-rich insert that includes a helix that blocks physical access to the T1 site. In crystalline form, the insert provides at least three additional Met-rich Cu(I) binding sites Cu5 (sCu), Cu6 and Cu7 that are proposed to facilitate rapid oxidation of bound Cu(I) to Cu(II) (S. K. Singh, et al., J. Biol. Chem., 2011, 43, 37849-37857). The activities of variants featuring mutations at sites Cu5 (D360M, M355LD360N), Cu6 (M358,362S), Cu7 (M364,368S) and Cu6,7 (M358,362,364,368S) were compared to that of the wild type form using three different air-stable model substrates (2,6-dimethoxyphenol, [Cu(I)(Bca)2](3-) and Cu(I)Cu(II)-PcoC, a periplasmic Cu(I) binding protein from E. coli). The results demonstrate that the three copper sites play related but distinct roles in CueO oxidase activities. The internal Cu5 site is part of the essential electron transfer pathway connecting surface-exposed sites Cu6 and Cu7 to site T1. Both Cu6 and Cu7 are dominant substrate-docking-oxidation (SDO) sites on the protein surface. However, under physiologically relevant conditions, the SDO function of Cu6 relies largely on an electron transfer pathway via Cu7 to Cu5. These Met-rich sites in CueO provide a robust cuprous oxidase function for control of Cu(I) toxicity. << Less

  Metallomics 7:776-785(2015) [PubMed] [EuropePMC]

• The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli.

  Outten F.W., Huffman D.L., Hale J.A., O'Halloran T.V.

  Copper is essential but can be toxic even at low concentrations. Coping with this duality requires multiple pathways to control intracellular copper availability. Three copper-inducible promoters, controlling expression of six copper tolerance genes, were recently identified in Escherichia coli. T ... >> More

  Copper is essential but can be toxic even at low concentrations. Coping with this duality requires multiple pathways to control intracellular copper availability. Three copper-inducible promoters, controlling expression of six copper tolerance genes, were recently identified in Escherichia coli. The cue system employs an inner membrane copper transporter, whereas the cus system includes a tripartite transporter spanning the entire cell envelope. Although cus is not essential for aerobic copper tolerance, we show here that a copper-sensitive phenotype can be observed when cus is inactivated in a cueR background. Furthermore, a clear copper-sensitive phenotype for the cus system is revealed in the absence of O(2). These results indicate that the cue pathway, which includes a copper exporter, CopA, and a periplasmic oxidase, CueO, is the primary aerobic system for copper tolerance. During anaerobic growth, however, copper toxicity increases, and the independent cus copper exporter is also necessary for full copper tolerance. We conclude that the cytosolic (CueR) and periplasmic (CusRS) sensor systems differentially regulate copper export systems in response to changes in copper and oxygen availability. These results underscore the increased toxicity of copper under anaerobic conditions and the complex adaptation of copper export in E. coli. << Less

  J. Biol. Chem. 276:30670-30677(2001) [PubMed] [EuropePMC]

• A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO.

  Roberts S.A., Wildner G.F., Grass G., Weichsel A., Ambrus A., Rensing C., Montfort W.R.

  CueO, a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present. The 1.7-A resolution structure of a crystal soaked in CuCl2 reveals a Cu(II) ion bound ... >> More

  CueO, a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present. The 1.7-A resolution structure of a crystal soaked in CuCl2 reveals a Cu(II) ion bound to the protein 7.5 A from the T1 copper site in a region rich in methionine residues. The trigonal bipyramidal coordination sphere is unusual, containing two methionine sulfur atoms, two aspartate carboxylate oxygen atoms, and a water molecule. Asp-439 both ligates the labile copper and hydrogen-bonds to His-443, which ligates the T1 copper. This arrangement may mediate electron transfer from substrates to the T1 copper. Mutation of residues bound to the labile copper results in loss of oxidase activity and of copper tolerance, confirming a regulatory role for this site. The methionine-rich portion of the protein, which is similar to that of other proteins involved in copper homeostasis, does not display additional copper binding. The type 3 copper atoms of the trinuclear cluster in the structure are bridged by a chloride ion that completes a square planar coordination sphere for the T2 copper atom but does not affect oxidase activity. << Less

  J. Biol. Chem. 278:31958-31963(2003) [PubMed] [EuropePMC]

• Oxidation of phenolate siderophores by the multicopper oxidase encoded by the Escherichia coli yacK gene.

  Kim C., Lorenz W.W., Hoopes J.T., Dean J.F.D.

  A gene (yacK) encoding a putative multicopper oxidase (MCO) was cloned from Escherichia coli, and the expressed enzyme was demonstrated to exhibit phenoloxidase and ferroxidase activities. The purified protein contained six copper atoms per polypeptide chain and displayed optical and electron para ... >> More

  A gene (yacK) encoding a putative multicopper oxidase (MCO) was cloned from Escherichia coli, and the expressed enzyme was demonstrated to exhibit phenoloxidase and ferroxidase activities. The purified protein contained six copper atoms per polypeptide chain and displayed optical and electron paramagnetic resonance (EPR) spectra consistent with the presence of type 1, type 2, and type 3 copper centers. The strong optical A(610) (E(610) = 10,890 M(-1) cm(-1)) and copper stoichiometry were taken as evidence that, similar to ceruloplasmin, the enzyme likely contains multiple type 1 copper centers. The addition of copper led to immediate and reversible changes in the optical and EPR spectra of the protein, as well as decreased thermal stability of the enzyme. Copper addition also stimulated both the phenoloxidase and ferroxidase activities of the enzyme, but the other metals tested had no effect. In the presence of added copper, the enzyme displayed significant activity against two of the phenolate siderophores utilized by E. coli for iron uptake, 2,3-dihydroxybenzoate and enterobactin, as well as 3-hydroxyanthranilate, an iron siderophore utilized by Saccharomyces cerevisiae. Oxidation of enterobactin produced a colored precipitate suggestive of the polymerization reactions that characterize microbial melanization processes. As oxidation should render the phenolate siderophores incapable of binding iron, yacK MCO activity could influence levels of free iron in the periplasm in response to copper concentration. This mechanism may explain, in part, how yacK MCO moderates the sensitivity of E. coli to copper. << Less

  J. Bacteriol. 183:4866-4875(2001) [PubMed] [EuropePMC]

• Cuprous oxidase activity of CueO from Escherichia coli.

  Singh S.K., Grass G., Rensing C., Montfort W.R.

  We have found CueO from Escherichia coli to have a robust cuprous oxidase activity, severalfold higher than any homologue. These data suggest that a functional role for CueO in protecting against copper toxicity in vivo includes the removal of Cu(I).

  J. Bacteriol. 186:7815-7817(2004) [PubMed] [EuropePMC]

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

  Djoko K.Y., Chong L.X., Wedd A.G., Xiao Z.

  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 he ... >> More

  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 Cu(II) (notation: Cu(II)-CueO). This was achieved in Mops buffer which has little affinity for Cu(2+). However, pH buffers that bind or precipitate Cu(2+) (Tris, BisTris, and KPi) generated enzyme with a vacant T4 site (notation: square-CueO) which has no phenol oxidase activity. Addition of excess Cu(2+) effectively generated a Cu(2+) 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): K(D) = 5.5 x 10(-9) M. CueO is involved in copper tolerance and has been suggested to be a cuprous oxidase. The anion [Cu(I)(Bca)(2)](3-) (Bca = bicinchoninate) acted as a novel chromophoric substrate. It is a robust reagent, being air-stable and having a Cu(I) affinity comparable to those of periplasmic Cu(I) binding proteins. The influences of pH buffer composition and of excess Cu(2+) on cuprous oxidation were diametrically opposite to those seen for phenol oxidation, suggesting that square-CueO, not Cu(II)-CueO, is the resting form of the cuprous oxidase. Steady-state kinetics demonstrated that the intact anion [Cu(I)(Bca)(2)](3-), not "free" Cu(+), is the substrate that donates Cu(I) 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 K(m) term consists of two components, allowing estimation of the transient affinity of T4 for Cu(I): K(D) = 1.3 x 10(-13) M. It may be concluded that Cu(I) carried by [Cu(I)(Bca)(2)](3-) is oxidized only upon complete transfer of Cu(I) 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. << Less

  J. Am. Chem. Soc. 132:2005-2015(2010) [PubMed] [EuropePMC]

• Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence.

  Singh S.K., Roberts S.A., McDevitt S.F., Weichsel A., Wildner G.F., Grass G.B., Rensing C., Montfort W.R.

  The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. H ... >> More

  The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Å with the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in k(cat) for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis. << Less

  J. Biol. Chem. 286:37849-37857(2011) [PubMed] [EuropePMC]