Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase

Abstract

Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen.

FIG. 1.

Spectroscopic analysis of recombinant T. vaginalis His-FDP. UV/VIS spectrum of pure His-FDP exhibits the typical features of flavoproteins. The inset shows the results of an SDS-PAGE analysis of purified recombinant His-FDP. Hom., bacterial lysate; FDP, purified His-FDP.

FIG. 2.

Amino acid sequence of TvFDP (XP001583562, TVAG_036010). The residues binding the diiron ligands are marked with an asterisk (*), and the conserved flavodoxin signature motif is shaded. The position of the hydrogenosomal processing peptidase cleavage site is marked with an arrow.

FIG. 3.

Subcellular localization of TvFDP. (A) SDS-PAGE of subcellular fractions of transformed T. vaginalis line overexpressing TvFDP (TvFDPWT); (B) SDS-PAGE of hydrogenosomes of untransformed T. vaginalis (TvT1); (C and D) Western blots probed with anti-TvFDP polyclonal antiserum. Molecular mass standards are indicated in kilodaltons. hom., homogenate; cyt., cytosol; hydr., hydrogenosomes.

FIG. 4.

Immunodetection of TvFDP in T. vaginalis cells. (A) Nomarski differential contrast; (B) visualization of malic enzyme, hydrogenosomal marker; (C) TvFDP labeling; (D) merge of color channels showing the presence of TvFDP in the hydrogenosomes with DAPI (4′,6′-diamidino-2-phenylindole) staining for nuclei.

FIG. 5.

Redox titration of the flavin cofactor monitored by visible spectroscopy at 460 nm. The full line was calculated for two consecutive one-electron redox processes, using molar absorptivities for the oxidized and semiquinone forms of 14,400 and 4,500, respectively, and with E1 = E2 = 25 mV.

FIG. 6.

Enzymatic reduction of TvFDP. Spectra of gradual reduction of His-FDP were obtained in an anaerobic system consisting of 44 mM pyruvate, 0.25 mM CoA, T.vaginalis PFOR (10 μg), and recombinant T. vaginalis ferredoxin (40 μg) in a phosphate buffer (100 mM KH₂PO₄/KOH, 150 mM NaCl, 10% glycerol [pH 7.4]). The dashed spectrum indicates reoxidized His-FDP after introduction of air into the cuvette. The recording time for one spectrum was 38 s.

FIG. 7.

EPR spectrum of as-purified TvFDP at 10 K. Microwave frequency, 9.39 GHz; microwave power, 2.0 mW; modulation amplitude, 1 mT.

FIG. 8.

Redox titration of the diiron center monitored by EPR spectroscopy, following the changes in EPR intensity at the g_med value of the diiron center. The full line was also calculated for two one-electron reduction processes and corresponds to the formation and disappearance of the EPR resonance of the Fe(III)-Fe(II) mixed valence state, with reduction potentials of 190 and 50 mV.