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Redundant and Flexible Electron Transfer Pathways Underlie MsrPQ-Mediated Repair of Oxidized Periplasmic Proteins
Laurent Loiseau1, Benjamin Ezraty1
1CNRS, Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Aix-Marseille University, Marseille, France.
Abstract:
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.
Insights
The MsrPQ system repairs oxidized proteins, crucial for bacterial stress resistance. This study finds ubiquinone is key, with other electron pathways providing backup for robust proteostasis.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- The MsrPQ system repairs oxidized methionine residues, vital for bacterial resistance to oxidative stress and maintaining envelope integrity.
- While ubiquinones were initially thought to be the primary electron source, in vitro studies suggested flavin reductase Fre and MsrQ's FMN cofactor are essential for electron transfer from cytoplasmic NADH to MsrP.
- The in vivo physiological relevance of Fre and FMN in the MsrPQ system remained unclear.
Purpose of the Study:
- To investigate the in vivo roles of the flavin reductase Fre and the FMN cofactor in the MsrPQ system using Escherichia coli.
- To determine the physiological relevance of Fre and FMN in bacterial oxidative stress response and protein repair mechanisms.
Main Methods:
- Genetic analysis involving deletion mutants (Δfre) and site-directed variants of MsrQ (MsrQH151A, MsrQR77A/R78A) in Escherichia coli.
- Phenotypic analysis including growth assays on methionine sulfoxide and colony morphology under anaerobic chlorate stress.
- Assessment of periplasmic protein redox states to evaluate MsrPQ system activity.
Main Results:
- Deletion of the fre gene did not impair MsrPQ activity, with Δfre mutants showing wild-type growth and normal colony morphology.
- Periplasmic protein redox states were unaffected in Δfre strains, unlike in ΔmsrPQ mutants.
- MsrQ variants lacking FMN or with altered quinone binding retained in vivo functionality, though with delayed growth phenotypes.
- Fre's necessity for MsrPQ activity increased with higher MsrQ expression or impaired ubiquinone binding.
Conclusions:
- The MsrPQ system utilizes a redundant network of electron transfer pathways, with ubiquinone as a central component.
- The contribution of Fre to MsrPQ function is conditionally dependent on MsrQ abundance and its quinone-binding capacity.
- This pathway redundancy ensures robust proteostasis, representing an evolutionary adaptation for bacterial survival under oxidative stress.
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