Most bacteria can generate ATP by respiratory metabolism, in which electrons are shuttled from reduced substrates to terminal electron acceptors, via quinone molecules like ubiquinone. Dioxygen (O₂) is the terminal electron acceptor of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone. Here, we characterize a novel, O₂-independent pathway for the biosynthesis of ubiquinone. This pathway relies on three proteins, UbiT (YhbT), UbiU (YhbU), and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory factor of the biosynthetic pathway, while UbiU and UbiV (UbiU-UbiV) are involved in hydroxylation reactions and represent a novel class of O₂-independent hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activity. The UbiT, -U, and -V proteins are found in alpha-, beta-, and gammaproteobacterial clades, including several human pathogens, supporting the widespread distribution of a previously unrecognized capacity to synthesize ubiquinone in the absence of O₂ Together, the O₂-dependent and O₂-independent ubiquinone biosynthesis pathways contribute to optimizing bacterial metabolism over the entire O₂ range.IMPORTANCE In order to colonize environments with large O₂ gradients or fluctuating O₂ levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence, and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes of Escherichia coli to this pathway and show that the pathway functions independently from O₂ In contrast, the long-described pathway for ubiquinone biosynthesis requires O₂ as a substrate. In fact, we find that many proteobacteria are equipped with the O₂-dependent and O₂-independent pathways, supporting that they are able to synthesize ubiquinone over the entire O₂ range. Overall, we propose that the novel O₂-independent pathway is part of the metabolic plasticity developed by proteobacteria to face various environmental O₂ levels.

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