Functional and biochemical characterization of [4Fe- 4S] ferredoxins mediating redox-network in Eubacterium callanderi KIST612

Abstract

Ferredoxin is a protein-based electron carrier that plays a pivotal role in the redox metabolism of acetogen. The types, numbers, and structural forms of ferredoxins encoded in acetogen genomes reflect their metabolic diversity and evolutionary specialization. This study aimed to identify and characterize key ferredoxins mediating electron transfer in the central metabolism of Eubacterium callanderi KIST612, a representative acetogen. Eleven ferredoxin candidates were screened from the E. callanderi KIST612 genome based on sequence homology and the presence of conserved Fer motifs, which form an α-antiparallel β- sandwich structure essential for [4Fe-4S] cluster stabilization. All candidates also contained the canonical cysteine arrangement (C-X2-C-X2-C-X3-C) responsible for coordinating the [4Fe-4S] cluster. Expression and spectroscopic analysis revealed expression and purification in the rest of the eleven candidate groups except for two. However, spectroscopic analysis revealed that only two candidates, ELI_1388 and ELI_3686, exhibited the characteristic absorption spectrum (strong absorption near 430 nm) of the [4Fe-4S] cluster, while the other nine showed negligible absorption, indicating insufficient cluster formation. Subsequently, enzyme assay with redox enzyme showed that ELI_1388 had a clear reactivity with CO dehydrogenase and formate dehydrogenase, indicating that it is involved in CO and formate-induced electron transfer within the Wood- Ljundahl pathway. ELI_3686, on the other hand, interacted strongly with [FeFe]-hydrogenase, implying a role in H2-dependent redox reactions and energy conservation. Together, these findings suggest that E. callanderi KIST612 possesses a functionally diversified ferredoxin in which individual ferredoxins participate in distinct metabolic routes, forming an integrated redox network that coordinates carbon and energy metabolism.