Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remain unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We identify ApbE flavinylation sites within structurally diverse protein domains and show that multi-flavinylated proteins, which may mediate longer distance electron transfer via multiple flavinylation sites, exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models.

This study explores the mechanisms bacteria use to transfer electrons outside the cytosol, a fundamental process involved in energy metabolism and environmental interactions. Central to this process is a phenomenon known as flavinylation, where a flavin molecule-a compound related to vitamin B2-is covalently attached to proteins, to enable electron transfer. We employed advanced genomic analysis and computational modeling to explore how this modification occurs across different bacterial species. Our findings uncover new types of proteins that undergo this modification and highlight the diversity and complexity of bacterial electron transfer mechanisms. This research broadens our understanding of bacterial physiology and informs potential biotechnological applications that rely on microbial electron transfer, including bioenergy production and bioremediation.