Ferredoxins are small iron-sulfur proteins that engage in one-electron transfer with oxidoreductases across all domains of life. The catalyzed reactions often include multiple electrons, , in the two-electron reduction of NADP during photosynthesis or the reduction of protons to H by the metalloenzyme hydrogenase. To date, the microscopic details of how ferredoxins facilitate multiple electron redox chemistry are unknown. Ferredoxins of the subfamily contain two [4Fe-4S] clusters, which allows for two one-electron transfer reactions. However, the iron-sulfur clusters of conventional 2[4Fe-4S]-type ferredoxins have very similar reduction potentials and conclusive evidence for the transfer of two electrons during a single protein-protein interaction (PPI) has not been reported. In this work, the electron transfer complexes between the clostridial 2[4Fe-4S] ferredoxin, Fd, and [FeFe]-hydrogenases from both (I) and (HydA), were investigated. Introducing a non-canonical amino acid near to one of the iron-sulfur clusters of Fd permitted the quantification of electric field changes the vibrational Stark effect by Fourier-transform infrared (FTIR) spectroscopy. Upon reduction, FTIR difference spectroscopy reported on protein structural changes and microscale thermophoresis revealed that the affinity between ferredoxin and hydrogenase is modulated by redox-dependent PPIs. Prompted by these findings, we suggest a model how ferredoxin efficiently facilitates multiple electron redox chemistry based on individual one-electron transfer reactions.