Flavodiiron proteins (FDPs) are enzymes identified in prokaryotes and a few pathogenic protozoa, which protect microorganisms by reducing O-2 to H2O and/or NO to N2O. Unlike most prokaryotic FDPs, the protozoan enzymes from the human pathogens Giardia intestinalis and Trichomonas vaginalis are selective towards O-2. UV/vis and EPR spectroscopy showed that, differently from the NO-consuming bacterial FDPs, the Giardia FDP contains an FMN with reduction potentials for the formation of the single and the two-electron reduced forms very close to each other (E-1 = -66 +/- 15 mV and E-2 = -83 +/- 15 mV), a condition favoring destabilization of the semiquinone radical. Giardia FDP contains also a non-heme diiron site with significantly up-shifted reduction potentials (E-1 = +163 20 mV and E-2 = +2 +/- 20 mV). These properties are common to the Trichomonas hydrogenosomal FDP, and likely reflect yet undetermined subtle structural differences in the protozoan FDPs. accounting for their marked O-2 specificity.