Biochemical characterization of a flavodiiron protein from bird parasite Histomonas meleagridis: superoxide as a reaction intermediate

Histomonas meleagridis is a parasitic protozoan which causes histomoniasis (blackhead disease) in a wide range of birds, including domesticated chickens and turkeys, representing a significant health problem in avian veterinary medicine. Despite being classified as an anaerobic parasite, H. meleagridis can survive transient exposure to oxygen (O₂) while little is known about the mechanisms that allow this organism to cope with exposure to varying O₂ levels. Inspecting the H. meleagridis genome revealed that this organism possesses two flavodiiron proteins (FDPs) belonging to classes A and F, which are known in other organisms to reduce O₂ to H₂O. The class F FDP found in H. meleagridis (HmFDPF) has domain organization previously described for similar proteins and contains a middle rubredoxin-like and C-terminal NADH:rubredoxin oxidoreductase domains in addition to an N-terminal FDP core. Here, we present extensive biochemical characterization of this protein to improve understanding of the coordinated activities of multiple redox centers that enable NADH-dependent reduction of O₂ to H₂O. We found that although superoxide is an HmFDPF reaction intermediate, only a very small amount of hydrogen peroxide (H₂O₂) is released as a byproduct. Moreover, HmFDPF does not possess NADH:H₂O₂ oxidoreductase activity and the enzyme is prone to quick inactivation by H₂O₂. In summary, our work expands our understanding of how microaerophilic protozoan parasites maintain their optimal redox state and minimize O₂ presence in their local environment, revealing mechanisms that can be exploited as drug targets against microaerophilic parasites.