MULTI-DOMAIN FLAVODIIRON PROTEINS FROM TRICHOMONAS VAGINALIS

BIROCCO, F.; IGLESIAS, ALBERTO A.; LIZÁRRAGA, A.; DE MIGUEL, N.; GUERRERO, SERGIO A.; ARIAS, DIEGO G.

Congreso; IX Congreso Internacional de Parasitología Neotropical; 2021

Trichomoniasis, caused by the extracellular parasite Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. Instead of mitochondria, T. vaginalis has hydrogenosomes that produce H2 as an end product of anaerobic energy metabolism and in which core enzymes, such as pyruvate-ferredoxin oxidoreductase and Fe?Fe hydrogenases, are highly oxygen-sensitive. However, the parasite typically experiences oxygen stress in its natural environment, during host transmission and hence must possess mechanisms to avoid the inactivation of oxygen-sensitive enzymes and to remove reactive oxygen species. Cytosolic NADH and NADPH oxidases are among the parasite?s most important O2 scavenging enzymes, and recently, a single-domain flavodiiron protein (FDP) with hydrogenosome localization was studied. The single-domain FDPs are enzymes with a minimal core of two domains: a metallo β-lactamase-like harboring a diiron center, and a flavodoxin, FMN containing domain. FDPs are O2 or NO reducing enzymes. They help many pathogens mitigate the NO produced by the immune system of the host and aid survival during fluctuating concentrations of oxygen. FDPs have a mosaic structure, being predicted to contain multiple extra domains. In this work, we present the functional characterization of two isoforms of multi-domain class-F FDP (TvNROR FDP1 and TvNROR-FDP2) with a modular arrangement. In silico analysis showed that these proteins have a canonical FDP domain and two extra domains, predicted to be a short rubredoxin-like and a NAD(P)H:rubredoxin oxidoreductase. TvNROR FDP1 and TvNROR-FDP2 were obtained by recombinant expression in Escherichia coli and purified proteins presented a dimeric structure by GF chromatography. The ability to bind prosthetic groups by the recombinant enzymes was evaluated by UV-Vis spectroscopy, TLC experiments, and metal quantification, showing evidence that TvNROR-FDPs conjugate FMN and/or FAD and iron atoms. Biochemical assays showed that both of them were able to catalyze the in vitro NAD(P)H-dependent O2 reduction. TvNROR-FDP1, but not TvNROR-FDP2, exhibited the NADH-dependent O2 partial-reduction to H2O2. For O2 reductase activity, even though the pH dependent activity profile resulted to be similar for both enzymes, a coenzyme-dependent profile was observed. In addition, the inactivation by DPI-Cl showed that flavin moiety is essential for the TvNROR-FDP activity. TvNROR-FDP1 showed the capability of electron transferring to one and two-electron-acceptor compounds, giving rise to diaphorase activity. However, the enzymes showed low activity for exogenous rubredoxin reduction. Basal expression was detected in T. vaginalis trophozoites by western blot experiment and their subcellular localization pattern revealed cytoplasmatic patterns. Our results suggest that TvNROR-FDP1/2 could be involved in oxidative stress protection in the parasite. To the best of our knowledge, this is the first characterization of class-F FDPs from an eukaryotic organism.Granted by ANPCyT (PICT2016-1778 and PICT2017-2268).