The flavodiiron protein from Syntrophomonas wolfei has five domains and acts both as an NADH:O or an NADH:H O oxidoreductase.

Flavodiiron proteins (FDPs) are a family of enzymes with a significant role in O /H O and/or NO detoxification through the reduction of these species to H O or N O, respectively. All FDPs contain a minimal catalytic unit of two identical subunits, each one having a metallo-β-lactamase-like domain harboring the catalytic diiron site, and a flavodoxin-like domain. However, more complex and diverse arrangements in terms of domains are found in this family, of which the class H enzymes are among the most complex. One of such FDPs is encoded in the genome of the anaerobic bacterium Syntrophomonas wolfei subsp. wolfei str. Goettingen G311. Besides the core domains, this protein is predicted to have three additional ones after the flavodoxin core domain: two short-chain rubredoxins and a NAD(P)H:rubredoxin oxidoreductase-like domain. This enzyme, FDP_H, was produced and characterized and the presence of the predicted cofactors was investigated by a set of biochemical and spectroscopic methodologies. Syntrophomonas wolfei FDP_H exhibited a remarkable O reduction activity with a k  = 52.0 ± 1.2 s and a negligible NO reduction activity (~ 100 times lower than with O ), with NADH as an electron donor, that is, it is an oxygen-selective FDP. In addition, this enzyme showed the highest turnover value for H O reduction (k  = 19.1 ± 2.2 s ) ever observed among FDPs. Kinetic studies of site-directed mutants of iron-binding cysteines at the two rubredoxin domains demonstrated the essential role of these centers since their absence leads to a significant decrease or even abolishment of O and H O reduction activities.