Abstract
A symmetric origin for bacterial ferredoxins was first proposed over 50 y ago, yet, to date, no functional symmetric molecule has been constructed. It is hypothesized that extant proteins have drifted from their symmetric roots via gene duplication followed by mutations. Phylogenetic analyses of extant ferredoxins support the independent evolution of N- and C-terminal sequences, thereby allowing consensus-based design of symmetric 4Fe-4S molecules. All designs bind two [4Fe-4S] clusters and exhibit strongly reducing midpoint potentials ranging from -405 to -515 mV. One of these constructs efficiently shuttles electrons through a designed metabolic pathway in Escherichia coli These finding establish that ferredoxins consisting of a symmetric core can be used as a platform to design novel electron transfer carriers for in vivo applications. Outer-shell asymmetry increases sequence space without compromising electron transfer functionality.