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J. Biol. Chem., Vol. 283, Issue 7, 4061-4068, February 15, 2008

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The O₂-scavenging Flavodiiron Protein in the Human Parasite Giardia intestinalis^*

From the Department of Biochemical Sciences, CNR Institute of Molecular Biology and Pathology and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza, University of Rome, Rome I-00185, Italy

The flavodiiron proteins (FDP) are widespread among strict or facultative anaerobic prokaryotes, where they are involved in the response to nitrosative and/or oxidative stress. Unexpectedly, FDPs were fairly recently identified in a restricted group of microaerobic protozoa, including Giardia intestinalis, the causative agent of the human infectious disease giardiasis. The FDP from Giardia was expressed, purified, and extensively characterized by x-ray crystallography, stopped-flow spectroscopy, respirometry, and NO amperometry. Contrary to flavorubredoxin, the FDP from Escherichia coli, the enzyme from Giardia has high O₂-reductase activity (>40 s^-1), but very low NO-reductase activity (0.2 s^-1); O₂ reacts with the reduced protein quite rapidly (milliseconds) and with high affinity (K_m 2 µM), producing H₂O. The three-dimensional structure of the oxidized protein determined at 1.9Å resolution shows remarkable similarities with prokaryotic FDPs. Consistent with HPLC analysis, the enzyme is a dimer of dimers with FMN and the non-heme di-iron site topologically close at the monomer-monomer interface. Unlike the FDP from Desulfovibrio gigas, the residue His-90 is a ligand of the di-iron site, in contrast with the proposal that ligation of this histidine is crucial for a preferential specificity for NO. We propose that in G. intestinalis the primary function of FDP is to efficiently scavenge O₂, allowing this microaerobic parasite to survive in the human small intestine, thus promoting its pathogenicity.

Received for publication, July 9, 2007 , and in revised form, October 9, 2007.

The atomic coordinates and structure factors (code 2Q9U) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by Ministero dell'Università e della Ricerca of Italy (PRIN Meccanismi molecolari e aspetti fisiopatologici dei sistemi bioenergetici di membrana (to P. S.) and FIRB RBLA03B3KC_004 (to M. B.)) and by Consiglio Nazionale delle Ricerche of Italy (CNR-GRICES joint project (to A. G.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1.

¹ To whom correspondence should be addressed: Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche c/o Dipartimento di Scienze Biochimiche, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185, Rome, Italy. Fax: 39-06-4440062; E-mail: alessandro.giuffre{at}uniroma1.it.

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