The cbb ₃ -type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase

Abstract

Copper (Cu)-containing proteins execute essential functions in prokaryotic and eukaryotic cells, but their biogenesis is challenged by high Cu toxicity and the preferential presence of Cu(II) under aerobic conditions, while Cu(I) is the preferred substrate for Cu chaperones and Cu-transport proteins. These proteins form a coordinated network that prevents Cu accumulation, which would lead to toxic effects such as Fenton-like reactions and mismetalation of other metalloproteins. Simultaneously, Cu-transport proteins and Cu chaperones sustain Cu(I) supply for cuproprotein biogenesis and are therefore essential for the biogenesis of Cu-containing proteins. In eukaryotes, Cu(I) is supplied for import and trafficking by cell-surface exposed metalloreductases, but specific cupric reductases have not been identified in bacteria. It was generally assumed that the reducing environment of the bacterial cytoplasm would suffice to provide sufficient Cu(I) for detoxification and cuproprotein synthesis. Here, we identify the proposed cbb3-type cytochrome c oxidase (cbb3-Cox) assembly factor CcoG as a cupric reductase that binds Cu via conserved cysteine motifs and contains 2 low-potential [4Fe-4S] clusters required for Cu(II) reduction. Deletion of ccoG or mutation of the cysteine residues results in defective cbb3-Cox assembly and Cu sensitivity. Furthermore, anaerobically purified CcoG catalyzes Cu(II) but not Fe(III) reduction in vitro using an artificial electron donor. Thus, CcoG is a bacterial cupric reductase and a founding member of a widespread class of enzymes that generate Cu(I) in the bacterial cytosol by using [4Fe-4S] clusters.

Copper tolerance in bacteria requires the activation of multiple accessory pathways

“…Cu(II) is more biologically inert, making it a relatively “safe” species for cells compared to Cu(I) (Rensing and Grass, 2003; Andreini et al ., 2008). Intracellular reductants, including respiratory chain complexes, enterobactin, and cysteine, contribute to the generation of Cu(I) from Cu(II) (Rodriguez‐Montelongo et al ., 1993; Rigo et al ., 2004; Grass et al ., 2004; Volentini et al ., 2011), and more intracellular Cu(I) is generated enzymatically for incorporation into copper‐containing cytochrome oxidases, at least in some organisms (Marckmann et al ., 2019). Copper toxicity is enhanced during anaerobic growth (Outten et al ., 2001; Espariz et al ., 2007; Tan et al ., 2017) and cells accumulate more copper under those conditions (Outten et al ., 2001; Macomber et al ., 2007).…”

Cu Homeostasis in Bacteria: The Ins and Outs

“…Despite the need for Cu(II) reduction in the bacterial cytosol, Cu-specific reductases have not been identified in bacteria until recently [ 157 ]. It was generally assumed that reducing agents, such as glutathione and ascorbic acid, are sufficient for providing enough Cu(I) for further transfer to Cu-chaperones [ 146 , 148 ].…”

“…The membrane-bound protein CcoG from the proteobacterium R. capsulatus was recently characterized as the founding member of a new and widespread class of cupric reductases in bacteria [ 157 ]. CcoG was initially identified as a putative assembly factor of cbb 3 -Cox , because it is encoded in the highly conserved ccoGHIS (also called fixGHIS ) gene cluster, located immediately downstream of the structural genes of cbb 3 -Cox.…”

“…In the absence of CcoG, cbb 3 -Cox activity and the Cu B -containing catalytic subunit CcoN are severely reduced. Furthermore, the absence of CcoG increases Cu-sensitivity, while its over-production promotes Cu tolerance [ 157 ]. These findings suggest that CcoG provides Cu(I) not only for cbb 3 -Cox assembly but also for Cu-chaperones, such as CopZ, and to P 1B -type ATPases for Cu(I) export to reduce Cu toxicity.…”

“…Homologues like YccM are smaller than CcoG as they lack the immunoglobulin (Ig)-like periplasmic domain at the C-terminus of CcoG. The function of the Ig-like domain in CcoG is currently unknown and requires further analyses [ 157 ].…”