Correlated Evolution of Symmetrical 4Fe–4 S Ferredoxin Binding Sites

Ferredoxins are ancient electron-transfer proteins containing two [4Fe–4 S] clusters that mediate electron transfer. While these clusters share a conserved structural framework, the extent to which their homologous cluster-binding environments exhibit coordinated evolutionary patterns remains unclear. Here, we performed a combined phylogenetic and sequence-based analysis of 250 bacterial dicluster 2[4Fe–4 S] ferredoxins. Phylogenetic analyses resolved four major clades, and correlations between the homologous cluster-binding environments of Cluster I and Cluster II differed among these lineages. The strongest coordinated sequence-level relationships were observed in simple ferredoxins from Clostridia , whereas weaker correlations were observed in insertion-bearing ferredoxins. Evolutionary conservation was further examined using the previously described TOTEMS framework (Fajardo et al. 2026), which demonstrated similar position-specific conservation patterns between the homologous cluster-binding environments of Cluster I and Cluster II. These findings are consistent with parallel sequence evolution around the two redox sites, suggesting that the homologous cluster-binding environments have evolved in a coordinated manner. Together, these results identify coordinated evolutionary patterns between homologous cluster-binding environments and provide a comparative framework for investigating sequence-level organization within bacterial dicluster ferredoxins.