Incorporation of iron into Tritrichomonas foetus cell compartments reveals ferredoxin as a major iron-binding protein in hydrogenosomes

1 Department of Parasitology, Faculty of Science, Charles University, Vini ná 7, 128 44, Prague 2, Czech Republic 2 Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 44, Prague 2, Czech Republic 3 Department of the Tropical Medicine, 1st Faculty of Medicine, Charles University, Faculty Hospital Bulovka, Studni kova 7, 128 00, Prague 2, Czech Republic 4 Johns Hopkins University, Bloomberg School of Public Health, W. Harry Feinstone Department of Molecular Microbiology and Immunology, 615 North Wolfe Street, Baltimore 21205, MD, USA Correspondence Jan Tachezy tachezy{at}natur.cuni.cz The intracellular transport of iron and its incorporation into organelles are poorly understood processes in eukaryotes and virtually unknown in parasitic protists. The transport of iron is of particular interest in trichomonads, which possess hydrogenosomes instead of mitochondria. The metabolic functions of hydrogenosomes, which contain a specific set of FeS proteins, entirely depend on iron acquisition. In this work the incorporation of iron into the cattle parasite Tritrichomonas foetus was monitored. Iron was efficiently taken up from 59 Fe-nitrilotriacetic acid and accumulated in the cytosol (88·9 %) and hydrogenosomes (4·7 % of the total radioactivity). Using atomic absorption spectrophotometry, an unusually high steady-state iron concentration in hydrogenosomes was determined [54·4±1·1 nmol Fe (mg protein) -1 ]. The concentration of iron in the cytosol was 13·4±0·5 nmol Fe (mg protein) -1 . Qualitative analysis of incorporated iron was performed using native gradient PAGE. The majority of the 59 Fe in the cytosol appeared as the labile-iron pool, which represents weakly bound iron associated with compounds of molecular mass ranging from 5000 to 30 000 Da. Ferritin was not observed in Tt. foetus , nor in two other anaerobic protists, Entamoeba histolytica and Giardia intestinalis . Analysis of Tt. foetus hydrogenosomes showed at least nine iron-binding compounds, which were absent in metronidazole-resistant mutants. The major iron-binding compound was identified as [2Fe–2S] ferredoxin of the adrenodoxin type. Abbreviations: DFO, desferrioxamine; LIP, labile-iron pool; NMML, nominal molecular mass (weight) limits; NTA, nitrilotriacetic acid; PFOR, pyruvate : ferredoxin oxidoreductase; ST, sucrose-Tris (buffer) The GenBank accession number for the sequence reported in this paper is AF545472 .