An iron-sulfur cluster as a new metal centre in a flavodiiron protein

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 predicted to have, besides the two core domains characteristic of this type of enzymes, an extra C- terminal domain putatively harbouring an iron-sulfur centre. Bioinformatic analyses showed that, thus far, Class E FDPs are only present in three other bacteria of the Syntrophomonas genus: Syntrophomonas palmitatica , Syntrophomonas zenhnderi and Thermosyntropha lipolytica. In this work, we extensively characterized the enzyme from Syntrophomonas wolfei (wild type, site directed mutants and truncated domains) and showed unequivocally, using EPR and Resonance Raman spectroscopies, that indeed it contains a [3Fe- 4S] ^1+/0 centre, a novelty in the field of FDPs. Structure prediction using Alphafold indicated some similarities to [3Fe4S] ^1+/0 containing ferredoxins. The reduction potentials of each cofactor were determined: +70 mV, -5/-70 mV and -90 mV for the FeS, diiron centre and flavin, respectively.