Oxidation of Phenolate Siderophores by the Multicopper Oxidase Encoded by theEscherichia coli yacKGene

Abstract

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 (⌭ 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.

A redox switch in CopC: An intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites

“…In the latter hypothesis, CopC and CopA may function together in copper resistance with CopA oxidizing Cu(I) bound to CopC to the less toxic Cu(II) form, as supported by genetic studies on the two homologues of these proteins from E. coli (47,48). Alternatively, copper delivered by CopC to CopA could catalyze the oxidase activity of CopA, as suggested by the copper-inducible oxidation of siderophores by CueO in E. coli (49).…”

In vitro studies indicate a quinone is involved in bacterial Mn(II) oxidation

“…In addition, fungal MCOs (laccases) (Hofer and Schlosser 1999;Schlosser and Hofer 2002) have been described that can oxidize Mn(II) to Mn (III). Some MCOs such as ceruloplasmin show a high degree of substrate specificity and others such as laccase possess a more relaxed substrate specificity oxidizing many different substrates including metals and phenolic compounds (Stoj and Kosman 2003) (Solano et al 2001) (Kim et al 2001). Fe(II) oxidation and fungal Mn(II) oxidation by MCOs serve as models for bacterially mediated Mn(II) oxidation.…”

A Labile Regulatory Copper Ion Lies Near the T1 Copper Site in the Multicopper Oxidase CueO

“…In general, multicopper oxidases couple four one-electron substrate oxidation steps to the four-electron reduction of dioxygen to water (14). CueO can oxidize a range of substrates in vitro, including catechols, siderophores, and Fe(II) (10), and this activity is greatly enhanced in the presence of excess (ϳ100 M) copper(II) ions (10,15), suggesting that binding of a labile copper is important for enzymatic activity. We recently determined the crystal structure of CueO at 1.4-Å resolution (15) and showed that its structure is similar to that of other multicopper oxidases, such as ascorbate oxidase (16) and several laccases (17)(18)(19).…”