The HoxEFUYH [NiFe]-hydrogenases from Synechocystis sp. PCC6803 catalyzes the activation of H2 coupled to ferredoxin or NAD(P)(H). The multimeric, bidirectional enzyme is comprised of a HoxEFU ferredoxin:NAD(P)H oxidoreductase diaphorase and a HoxYH [NiFe]-hydrogenase subcomplex that catalyzes reversible H2 oxidation. A goal of our research is to understand how the HoxEFU diaphorase orchestrates electron transfer through its complex network of FeS clusters to effectively link pyridine oxidation/reduction to ferredoxin reactivity.1 Deciphering how electron transfer is controlled is challenged by the cofactor rich content of the diaphorase, which includes three [2Fe-2S] clusters, four [4Fe-4S] clusters, and a flavin cofactor. To address this and determine how properties of the cluster network are tuned to mediate multi electron exchange reactions, we have been using a combination of electron paramagnetic resonance (EPR) spectroscopy, structural approaches, and novel light-driven methods. Our results suggest a possible redox-dependent gating mechanism for linking pyridine oxidation/reduction to ferredoxin reactivity.2 This is substantiated by observed changes in the redox potential of HydE, which can vary by more than 100 mV when either isolated or in the HoxEFU complex. Preliminary single-particle cryo-EM reconstruction of HoxEFU further shows the structure is a dimer of HoxEFU monomers, where HoxU forms at the dimer interface. Together, the results are informing on how electron transfer is modulated in multi-cluster systems that function as part of complex energy transduction networks, where efficiency and directionality are highly desired properties.