Redundant and Flexible Electron Transfer Pathways Underlie MsrPQ-Mediated Repair of Oxidized Periplasmic Proteins.

The MsrPQ system is essential for the repair of oxidized methionine residues in periplasmic proteins, ensuring bacterial resistance to oxidative stress and envelope integrity. While ubiquinones were initially proposed as the primary electron source for MsrPQ, in vitro studies suggested that the flavin reductase Fre and the FMN cofactor of MsrQ are essential for electron transfer from cytoplasmic NADH to MsrP. However, their physiological relevance in vivo remained unclear. Here, we investigated the role of Fre and FMN in the MsrPQ system using Escherichia coli as a model organism. We demonstrate that deletion of the fre gene does not impair MsrPQ activity, as Δfre mutants exhibit wild-type growth on methionine sulfoxide and maintain normal colony morphology under anaerobic chlorate stress. Additionally, the redox state of periplasmic proteins remains unaffected in Δfre strains, unlike in ΔmsrPQ mutants where these proteins accumulate in an oxidized form. Furthermore, we show that the MsrQH151A and MsrQR77A/R78A variants, which lack the FMN cofactor and exhibit altered quinone-binding capacity, respectively, retain full functionality in vivo, albeit with a delayed growth phenotype on methionine sulfoxide. However, we show that Fre becomes necessary for efficient methionine sulfoxide utilization by the MsrPQ system when MsrQ is plasmid-expressed, and that this dependence is further increased when MsrQ is impaired in ubiquinone binding. These observations indicate that the contribution of Fre is conditionally determined by MsrQ abundance. Collectively, our findings reveal that the MsrPQ system operates through a redundant network of electron transfer pathways, with ubiquinone as a central player. This redundancy likely represents an evolutionary adaptation to ensure robust proteostasis even when specific components of the electron transfer chain are compromised.