The flavodiiron protein from <i>Syntrophomonas wolfei</i> has five domains and acts both as an NADH:O<sub>2</sub> or an NADH:H<sub>2</sub>O<sub>2</sub> oxidoreductase
Maria C. Martins, C. M. Alves, Miguel Teixeira, Filipe Folgosa
Abstract
Flavodiiron proteins (FDPs) are a family of enzymes with a significant role in O 2 /H 2 O 2 and/or NO detoxification through the reduction of these species to H 2 O or N 2 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 2 reduction activity with a k cat = 52.0 ± 1.2 s −1 and a negligible NO reduction activity (~ 100 times lower than with O 2 ), 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 2 O 2 reduction ( k cat = 19.1 ± 2.2 s −1 ) 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 2 and H 2 O 2 reduction activities.