Tuning the Electron Transfer Activity of a 2Fe2S Ferredoxin Using Homologous Recombination and an Escherichia coli Selection

Iron-sulfur (Fe-S) cluster containing ferredoxins (Fd) are low potential (high energy) metalloproteins that distribute electrons across diverse metabolic pathways by leveraging interactions with an array of partner proteins (the Fd interactome). In vitro studies have revealed Fd paralogs with high sequence identity (>90%) can vary drastically in their functional profiles when coupling to the same e- donors and acceptors. However, these studies have not yet provided sufficient insight into Fd to tune their redox proportioning to different partner proteins for synthetic biology applications. To improve our understanding of the underlying sequence-structure-function relationships that dictate Fd redox proportioning and cycling efficiencies, we are recombining Mastigocladus laminosus and Prochlorococcus cyanomyophage P-SSM2 Fd, plant-type 2Fe2S Fd that display ~53% sequence identity, and analyzing the cellular functions of chimeras. Ferredoxin function is being quantified by analyzing the complementation of an Escherichia coli sulfide auxotroph, which requires electron transfer through a linear redox pathway that is made up of Fd-NADP⁺ reductase, Fd, and sulfite reductase. To quantify the relative activity of chimeras, Fd expression was placed under control of the promoter P_LtetO-1 and bacterial complementation has been measured in the presence of varying anhydrotetracyline (aTc) concentrations. Our preliminary experiments have revealed that some chimeras require low levels of aTc (2.5 ng/mL) to complement bacterial growth half maximally, like M. laminosus Fd, while other chimeras only partially complement bacterial growth at high levels of aTc (250 ng/mL). Ongoing efforts are investigating whether these differences in cellular activity arise because of changes in cellular expression, protein stability, cofactor binding, redox potential, partner binding, and partner allosteric interactions. By comparing the complementation of dozens of Fd, our results will allow us to calibrate how Fd redox activity relates to structural disruption calculated using biophysical models. In addition, they will provide fundamental insight into Fd sequence-structure-function relationships, which are currently needed to inform the construction of orthogonal redox circuits that can operate alongside host cell redox circuits.