Iron-sulfur cluster containing ferredoxins (Fds) are ancient proteins found in all three branches of life that participate in diverse energy transduction pathways including those found within organisms that are most closely related to the first free-living ancestors of Bacteria and Archaea (methanogens, acetogens, and sulfur-reducing microbes). Many researchers in the Fe-S field propose that modern clostridial-type 4Fe-4S Fds arose through duplication, fusion, and mutation of smaller peptides that also coordinated Fe-S clusters and supported ancient energy transduction pathways. The biophysical properties of small bio-inspired peptides that coordinate Fe-S clusters have been widely studied in vitro and found to display redox potentials that are compatible with energy transduction pathways essential for life, such as sulfite reduction to sulfide. However, fossil-like peptides have not yet been assayed for their ability to support any energy transduction pathways within cells. It remains unclear which of the many possible Fe-S-binding peptides can fulfill the functions of modern Fds within living cells, and how the sequence (length, cysteine motifs), physical (stability, redox potential) and functional (binding selectivity) properties of active Fe-S-binding peptides differ from modern Fds that relay redox for diverse reactions within cells. We hypothesize that the best way to understand fossil-like Fe-S-binding peptides that supported ancient energy transduction pathways is to create libraries of fragmented Fds having various peptide lengths, use cellular growth selections to mine these libraries for peptides that are redox active in cells and capable of supporting energy transduction, and sequence those variants that are active within the context of the complex cellular milieu. By analyzing the sequence, structure, cofactors, redox potentials, and oligomeric states of peptides selected from these libraries, we propose to obtain direct evidence supporting a role for Fe-S-binding peptides in early evolution within cells and establish the characteristics of peptides that are capable of supporting a cellular energy transduction reaction. Our specific objectives are to: (i) identify modern Fds that support energy transduction processes within cells by assaying activities using simple bacterial growth selections, (ii) find minimal Fe-S-binding peptides that support energy transduction within cells by fragmenting Fds and selecting for active peptides, and (iii) determine if the Fe-S-binding peptides are less selective than natural Fds in their ability to support different energy transduction reactions. The results from the proposed studies will be relevant to the Exobiology program and its interest in understanding the Early Evolution of Life because this research will identify putative primordial peptides that are capable of serving as redox cofactors for energy transduction within cells, establish the minimal peptide features within Fds that are required to coordinate an Fe-S cluster and support energy transduction within cells, and elucidate the biochemical and biophysical properties of active Fe-S-binding peptides (e.g., Fe-S cluster type, oligomeric state, redox potentials, and binding selectivity). These studies will also yield multiple Fd-derived fragments and Fds that support sulfite reduction and allow comparison of the sequence-structure-redox properties of fossil-like peptides and modern Fds in the same context. Furthermore, these studies will yield evidence that Fe-S peptides could support biotic S isotope fractionation signatures observed in putative molecular fossils from the Archaean, provide a framework for future studies of electron transfer among ancestral proteins involved in early energetic processes and their evolution, and facilitate future analysis of prebiotic chemical reactions supported by Fe-S peptides and allow comparisons with pure minerals (e.g., pyrite) and modern Fds.