title: Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo.
abstract: Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.
authors.affiliation: Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Pandini
authors.forename: Gianluca
authors.initials: G
authors.lastname: Caprini
authors.forename: Nadine
authors.initials: N
authors.lastname: Thomsen
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aliverti
authors.forename: Frank
authors.initials: F
authors.lastname: Seeber
authors.forename: Giuliana
authors.initials: G
authors.lastname: Zanetti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000378:metabolism*
chemical_list: D005288:Ferredoxins; D005287:Ferredoxin-NADP Reductase