# Unraveling the pathway of copper delivery to cytochrome c oxidases in the free-living bacterium Caulobacter vibrioides

## TL;DR

This study explores how the bacterium Caulobacter vibrioides delivers copper to its cytochrome c oxidases, revealing a unique pathway involving a novel outer membrane protein.

## Contribution

The discovery of a novel outer membrane TonB-dependent receptor (TccA) involved in copper delivery to aa3-Cox in C. vibrioides.

## Key findings

- C. vibrioides uses a FixI-type Cu transporter and PccA for cbb3-Cox activity.
- A novel outer membrane receptor TccA is required for aa3-Cox function.
- cbb3-Cox is upregulated under microaerobic conditions, while aa3-Cox dominates under normoxic conditions.

## Abstract

Copper (Cu) is an essential micronutrient that serves as a cofactor for many enzymes but becomes toxic when present in excess. In most bacteria, CopA-like P1B-type ATPases mediate Cu detoxification by exporting cytoplasmic Cu to the periplasm or extracellular environment. In this study, we show that Caulobacter vibrioides lacks a canonical CopA-like ATPase but encodes a single FixI/CcoI-type Cu-transporting ATPase, previously implicated in Cu delivery to the cbb3-type cytochrome c oxidase (Cox) in species such as Rhodobacter capsulatus. C. vibrioides harbors two terminal cytochrome c oxidases in its cytoplasmic membrane: an aa3-type and a cbb3-type Cox. We also demonstrate that the activity of cbb3-Cox requires the FixI-type Cu transporter and the periplasmic Cu chaperone PccA. In contrast, aa3-Cox activity depends on PccA and the inner membrane-bound protein CtaG. Since the mechanism of Cu acquisition for aa3-Cox remains largely unknown, we conducted a genetic screen and identified a novel outer membrane TonB-dependent receptor (TccA) that is specifically required for aa3-Cox function. We also showed that cbb3-Cox is upregulated under microaerobic conditions, possibly such as those encountered on solid media where O2 diffusion is limited. Under normoxic conditions, the expression and the activity of cbb3-Cox decrease, and aa3-Cox becomes the predominant terminal oxidase. These findings demonstrate that C. vibrioides differentially utilizes its Cox enzymes in response to O2 availability and relies on a distinct Cu trafficking pathway for their maturation, including an outer membrane component that has not been previously described in bacterial Cu homeostasis.