Fe-S cluster biogenesis is an essential pathway coordinated by a network of protein-protein interactions whose functions include desulfurase activity, substrate delivery, electron transfer and product transfer. In an effort to understand the intricacies of the pathway, we have developed an in vitro assay to follow the ferredoxin role in electron transfer during Fe-S cluster assembly. Previously, assays have relied upon the non-physiological reducing agents dithionite and dithiothreitol to assess function. We have addressed this shortcoming by using electron transfer between NADPH and ferredoxin-NADP-reductase to reduce ferredoxin. Our results show that this trio of electron transfer partners are sufficient to sustain the reaction in in vitro studies, albeit with a rate slower compared with DTT-mediated cluster assembly. We also show that, despite overlapping with the CyaY protein in binding to IscS, Fdx does not interfere with the inhibitory activity of this protein. We suggest explanations for these observations which have important consequences for understanding the mechanism of cluster formation. Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. (C) 2015 The Authors. Published by Elsevier B.V.