Syntrophomonas wolfei contains two distinct multiple domain flavodiiron proteins (FDPs) of Classes H and E, presumably acting as oxygen reductases to protect this anaerobic bacterium from oxidative stress due to exposure to environments containing, even if only transiently, oxygen. The Class E FDP was previously predicted by us to have, besides the two core domains characteristic of this type of enzymes, an extra C-terminal domain putatively harboring an iron-sulfur center. This C-terminal domain is exclusive to this class of FDPs and has homology with a protein domain family "Fer4_19" which may contain a [3Fe-4S](1+/0) or a [4Fe-4S](2+/1+) cluster. In this work, we extensively characterized the enzyme from S. wolfei (wild type, site-directed mutants, and truncated iron-sulfur domain) and showed unequivocally, using EPR and Resonance Raman spectroscopies, that indeed it contains a [3Fe-4S](1+/0) center, a novelty in the field of FDPs. Structure prediction using Alphafold2 indicated some similarities of the FeS domain to [3Fe-4S](1+/0) containing ferredoxins. The identification of this new type of redox center associated with an FDP could represent the first step towards identifying a novel electron transfer chain within this protein family. Additionally, the spectroscopic characterization of the FMN from the flavodoxin-like domain suggests that the semiquinone form is the active reduced state of this flavin cofactor. Furthermore, the presence of a minor species possibly associated with the flavin moiety was identified, displaying a so far undescribed UV-visible spectrum.