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

Abstract

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu1+ to the less toxic Cu2+. Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb3-type cytochrome oxidase (cbb3-Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb3-Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF.

Posttranscriptional Regulation by Copper with a New Upstream Open Reading Frame

“…There are preliminary indications that other families of uORFs might posttranscriptionally regulate genes coding for copper export systems in response to the metal ( 61 – 63 ). In a recent work, the activity of a multicopper oxidase, CutO, was shown to depend on an upstream ORF with similarities to CruR, CutF, which the authors conjectured works as a Cu chaperone ( 64 ). It is tempting to speculate that CutF may also be an uORF that activates the expression of its downstream genes in response to copper.…”

Metabolic Sensing of Extra-cytoplasmic Copper Availability via Translational Control by a Nascent Exported Protein

“…Starting with our previous rule-based list (list A, 317 entries, Fig. 6A) of CutF-like proteins encoded by genes neighboring cutO (15), an iterative search with jackhammer against reference proteomes in the UniProt database (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…This ensures sufficient Cu supply for cuproprotein biogenesis while preventing accumulation of free Cu (4,(9)(10)(11)(12). Periplasmic multi-copper oxidases provide a further defense line against Cu toxicity by converting Cu(I) to the less toxic Cu(II) (13)(14)(15).…”

“…Bioinformatic searches identified CutF as a member of the DUF2946-like family of proteins, which are abundant in Pseudomonadota and are often encoded upstream of cuproenzymes, periplasmic Cu chaperones, and Cu transporters (15). Despite their abundance, the function of CutF-like proteins is unknown.…”

“…One example is the proteolysis of CueR by the AAA + proteases Lon, ClpXP and ClpAP (23). Another example is the multi-copper oxidase CutO in Rhodobacter capsulatus (15, 24). In response to Cu, production of CutO is regulated by an mRNA stem-loop sequence (SL) located between cutF and cutO and strictly requires the presence of an intact cutF in the tricistronic cutFOG operon ( Fig.…”