The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster

Abstract

CopG is an uncharacterized protein ubiquitous in gram-negative bacteria whose gene frequently occurs in clusters of copper-resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa.Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system.

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product

“…It is frequently present in Cu resistance-conferring gene clusters encoding CueO, CopA, and CusCBA ( Monchy et al, 2006 ; Wiethaus et al, 2006 ; Behlau et al, 2011 ; Marrero et al, 2012 ). P. aeruginosa CopG contains a cysteine-bridged tetranuclear Cu cluster and contributes to Cu resistance under anaerobic conditions via its Cu oxidoreductase activity ( Hausrath et al, 2020 ). In addition to its enzymatic activity, CopG is proposed to be involved in Cu transfer reactions ( Hausrath et al, 2020 ).…”

The YcnI protein from Bacillus subtilis contains a copper-binding domain

“…4 F ) ( 42 ), and in the MopE protein, an oxidized tryptophan residue serves as a metal ligand ( 43 ). A recent study of CopG, a copper-dependent oxidoreductase, also features a strictly conserved tryptophan residue in close proximity to one of the metal ions ( 44 ). The orientation of the tryptophan in YcnI is most similar to that of the tryptophan in the Cu-binding site of CusF.…”

Cu Homeostasis in Bacteria: The Ins and Outs

“…Periplasmic CopG-like proteins are widely distributed in Gram-negative bacteria and also found genetically linked to the copA and cusCBA Cu export systems. They contribute to Cu resistance under anaerobic conditions and consist of a thioredoxin-like domain that binds a tetranuclear Cu cluster via a conserved CxCC motif and two adjacent methionine residues [ 346 ] ( Figure 10 ). It has been proposed that CopG oxidizes Cu(I) to Cu(II), and also transfers Cu to other cuproproteins, such as CueO [ 346 ].…”

“…They contribute to Cu resistance under anaerobic conditions and consist of a thioredoxin-like domain that binds a tetranuclear Cu cluster via a conserved CxCC motif and two adjacent methionine residues [ 346 ] ( Figure 10 ). It has been proposed that CopG oxidizes Cu(I) to Cu(II), and also transfers Cu to other cuproproteins, such as CueO [ 346 ]. In R. capsulatus , cutG ( rcc02109 ) is part of the cutFOG operon [ 254 ].…”