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

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 (Km ≤ 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.

The flavodiiron proteins (FDP, 2 originally named A-type flavoproteins (1)) are widespread among Bacteria and Archaea, either strict or facultative anaerobes, where they have been proposed to play a role in the response to nitrosative and/or oxidative stress (2,3). A few prokaryotic FDPs have been characterized to date, namely those from the bacteria Desulfovibrio gigas (originally named rubredoxin:oxygen oxidoreductase, ROO (4 -7), and hereafter denoted FDP Dg ), Escherichia coli (named flavorubredoxin, FlRd, 3 Refs. 2, 8 -11), Desulfovibrio vulgaris (12), Moorella thermoacetica (FDP Mt , (13,14)), and the homologous enzyme from the methanogenic archaeon Methanothermobacter marburgensis (FDP Mm , Refs. 15,16). The FDPs contain two redox centers: a FMN, the electron entry site into the enzyme, and a non-heme Fe-Fe center, the active site (13). They are cyanide-insensitive enzymes able to catalyze the reduction of O 2 (to H 2 O) and/or NO (to N 2 O). Some of these enzymes are almost exclusively reactive toward NO (such as E. coli FlRd, Refs. 2,9), 4 others toward O 2 (such as the M. marburgensis enzyme, (15)), whereas some FDPs catalyze the reduction of both gases, though with different efficiency (7,12,13). These enzymes are expected to play a protective role in anaerobic or microaerobic microorganisms that need to survive under O 2 and cope with NO produced by the host defense system to counteract infection (17,18).
Surprisingly, a few years ago, genes coding for FDPs were identified also in the genome of a few eukaryotes, namely some amitochondriate microaerobic protozoan parasites including Giardia intestinalis, Trichomonas vaginalis, Spironucleus barkhanus, Mastigamoeba balamuthi, and several Entamoeba strains (19 -22). Despite their potential pathophysiological relevance, structural and functional information on eukaryotic FDPs is still marginal.
G. intestinalis is the causative agent of giardiasis, a widespread intestinal infectious disease in humans (23). Very recently, its genome has been completely sequenced (24), lead-* 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 atomic coordinates and structure factors ( ing to conclude that Giardia is an early diverging protozoon with very simplified metabolic pathways. Although Giardia has a relatively poor tolerance to O 2 , it preferentially colonizes the fairly aerobic upper part of the small intestine (duodenum and jejunum). The parasite has an essentially fermentative energy metabolism (25). It lacks the conventional respiratory oxidases as well as the systems (catalase, superoxide dismutase, glutathione reductase) responsible for the scavenging of radical oxygen species (ROS) (26).
Here we present the three-dimensional structure of the FDP from Giardia solved by x-ray crystallography, and provide evidence that in addition to the H 2 O-producing NADH-oxidase previously characterized (27), Giardia employs the FDP system to efficiently cope with O 2 . To the best of our knowledge this is the first eukaryotic FDP characterized, to date.

EXPERIMENTAL PROCEDURES
Materials-Stock solutions of ϳ2 mM NO (Air Liquide, France) were prepared by equilibrating degassed water with the pure gas at 1 atm and room temperature. The concentration of E. coli FlRd-red and of the genetically truncated rubredoxin domain of E. coli flavorubredoxin (Rd) in the oxidized state was determined using ⑀ 455 nm ϭ 12 mM Ϫ1 cm Ϫ1 and ⑀ 484 nm ϭ 7 mM Ϫ1 cm Ϫ1 , respectively.
Cloning, Expression, and Purification-The gene coding for the G. intestinalis FDP (FDP Gi ) 5 , synthesized by GENEART GmbH (Regensubrg, Germany), was cloned in pET28b(ϩ) vector (Novagene). Expression of the His-tagged protein in E. coli BL21-Gold (DE3) cells was induced with 0.1 mM isopropyl ␣-Dthiogalactoside after supplementing the medium with 100 M ferrous ammonium sulfate (NH 4 Fe(SO 4 ) 2 ⅐6H 2 O); cells were grown at 25°C in M9 medium. The recombinant protein was purified by Nickel affinity chromatography, followed by gel filtration chromatography to remove excess imidazole. After His tag cleavage with thrombin, the protein was subjected to a second step of nickel affinity and gel filtration chromatography.
Protein Characterization-The protein concentration was determined by the Bicinchoninic Acid Assay (BCA) (28). The FMN was quantitated according to Ref. 29 and iron using the ferrozine assay (30) (see supplemental materials for details). The quaternary structure of FDP Gi in solution was determined using a Tricorn TM Superdex 200 -10/300 HPLC column (Amersham Biosciences GE Healthcare). Stopped-flow experiments were carried out with a thermostated instrument (DX.17MV, Applied Photophysics, Leatherhead, UK) equipped with a diode-array (light path, 1 cm). Time-resolved absorption spectra were recorded with an acquisition time of 2.5 ms per spectrum. Kinetic data were analyzed by nonlinear leastsquares regression analysis using the software MATLAB (MathWorks, South Natick, NA). Nitric oxide (NO) and oxygen consumption measurements were carried out using Clark-type selective electrodes (Apollo 4000 from World Precision Instruments or Oxygraph-2k from Orobors Instruments). In these measurements, turnover numbers were estimated based on the amount of the enzyme incorporating FMN.
Protein Crystallization-Best crystals of FDP Gi were obtained by hanging drop vapor diffusion method using the following reservoir solution: 0.1 M sodium acetate pH 4.6, 14 -16% PEG 3350, and 0.2 M potassium nitrate. Drops were prepared by mixing 1.0 l of reservoir solution and 1.0 l of protein (12 mg/ml) and allowed to equilibrate against 0.5 ml of the reservoir. Crystals were cryoprotected in 0.1 M sodium acetate pH 4.6, 20% PEG 3350, 0.4 M potassium nitrate and 20% PEG 200. Diffraction data were collected at 100 K at the ID29 beamline of the ESRF Synchrotron (Grenoble, France). Data were indexed and integrated with DENZO and SCALEPACK, respectively (31). The best quality crystal diffracted up to 1.9 Å resolution (Table 1). Two molecules were found in the asymmetric unit corresponding to a solvent content of 45.4% and a V M coefficient of 2.3.
Crystal Structure Determination and Refinement-The crystal structure of FDP Gi was determined by Molecular Replacement with MOLREP (32) using the structure of the FDP from M. thermoacetica (PDB code 1YCF, Ref. 14) as template. A rigid body refinement with REFMAC5 (33), followed by ARP/wARP (procedure: improvement of maps by atom update and refinement), was then applied to improve the quality of the initial map. The model was built with COOT (34), iteratively refined using REFMAC5 (33), visually inspected, and manually rebuilt. Solvent molecules were added into the F O -F C density map. The iron atoms, the oxo (hydroxo or aquo) bridges and the nitrate ions were introduced only in the late stages of the refinement to prevent model bias. The final model was refined to R factor and R free equal to 16.6 and 20.4%, respectively, at 1.9-Å resolution ( Table 1).
The FDP Gi crystal structure consists of a total of 814 residues (residues 4 -412 for monomer A and residues 6 -412 for monomer B), two FMN, four Fe atoms, two oxo (hydroxo or aquo) bridges, five nitrate ions, and a total of 781 water molecules in the asymmetric unit. Residues 4, 124, 376, 387, 408, 412 of monomer A and residues 124, 216, 376, and 412 for monomer B 5 GenBank TM accession code 27981644.

The Flavodiiron Protein from Giardia
were fitted as alanine because of the lack of density for the side chain. The refined model was checked for geometrical quality by using PROCHECK (35). Ramachandran statistics show that 99.5% of the residues lie in allowed regions, while four residues (Asp-55 and Phe-382 in both monomers) lie in disallowed regions. Asp-55 is in the proximity of the Fe-Fe center, while Phe-382 contacts FMN. The residues topologically corresponding to Asp-55 and Phe-382 lie in disallowed regions of the Ramachandran plot also in FDP Mt and in generously allowed regions in FDP Mm . Structural superposition was performed using SSM Superposition as implemented in COOT (36). Analysis of ligand-protein contacts was performed with SPACE (37

General Properties of the Purified Protein
The recombinant FDP from Giardia (FDP Gi ) was purified to homogeneity with a typical yield of ϳ3 mg of protein per gram of E. coli cells. SDS-PAGE shows that the protein is a single polypeptide with molecular mass ϳ45 kDa, consistent with the value of 46,904 Da calculated from the amino acid sequence. HPLC analysis (not shown) indicates that in solution the protein is a tetramer with an apparent molecular mass of 169 Ϯ 17 kDa. As purified, each monomer of FDP Gi contains ϳ1.5 Fe and ϳ0.5 FMN (see supplemental materials), instead of 2 Fe and 1 FMN, which implies partial loss of the cofactors during purification or incomplete incorporation during expression. The oxidized protein displays a UV/visible absorption spectrum dominated by the flavin cofactor, with bands at 358 and 461 nm. In the presence of NADH, only upon addition of catalytic concentrations of the E. coli proteins FlRd-red and Rd, FDP Gi is reduced and absorption in the visible region almost completely bleached (see dashed spectra in Fig. 1).

The Function
Kinetics of Oxidation by O 2 and NO-The reactivity of reduced FDP Gi with O 2 and NO was tested at 20°C by stoppedflow absorption spectroscopy. As shown in Fig. 1 (top panel) the reduced protein is very rapidly oxidized even by a 2-fold excess of O 2 , suggestive of a high affinity; ϳ80% of the protein is oxidized with a t1 ⁄ 2 ϳ3 ms, the remained being oxidized on a much longer time scale (a few seconds). As the stopped-flow instrument collects spectra every 2.5 ms, the fast oxidation of the large majority of the protein is at the limits of the time resolution of the technique, proceeding at an estimated second order rate constant k Ͼ 10 7 M Ϫ1 s Ϫ1 . The protein is instead oxidized by H 2 O 2 much more slowly (k ϭ 1.7 s Ϫ1 at [H 2 O 2 ] ϭ 60 M, not shown), ruling out a peroxidatic activity for FDP Gi .
Compared with O 2 , the protein is much less reactive toward NO, being oxidized only within seconds even at very high NO concentrations. For instance, at [NO] ϭ 650 M, the oxidation of FDP Gi , followed under strictly anaerobic conditions, proceeds at k ϭ 2.7 s Ϫ1 (Fig. 1, bottom panel), the reaction being even slower at lower NO concentrations. Under anaerobic conditions, low molecular weight nitrosothiols, such as nitrosocysteine, can also oxidize FDP Gi , although at very low rate (k ϭ 0.1 s Ϫ1 at 50 M nitroso-cysteine, not shown).
O 2 and NO Amperometric Measurements-The ability of FDP Gi to catalyze the consumption of O 2 or NO was tested by using selective electrodes. In these assays, a large excess of NADH (1 mM) was used as the primary electron donor, and the E. coli proteins FlRd-red and Rd were added to shuttle electrons to FDP Gi . As shown in Fig. 2 Fig. 2, top panel); as expected, the activity vanishes upon FDP Gi denaturation, as well as if the reducing substrate, Rd, is omitted (not shown). Addition of catalase has no effect on the apparent rate of O 2 consumption, pointing to H 2 O (and not H 2 O 2 ) as the reaction product: consistently, by monitoring NADH oxidation at 340 nm in control experiments, we measured a NADH/O 2 stoichiometry equal to 2 (not shown). As shown in Fig. 2 (top panel),   (12,13), no irreversible inactivation of the Giardia enzyme is observed during turnover with O 2 , unless catalase is omitted in the assay; in the latter case, H 2 O 2 produced by the reaction of FlRd-red with O 2 slowly, but progressively inhibits the enzyme (not shown).
Similar experiments have been carried out to test also the NO-reductase activity of FDP Gi under anaerobic conditions (Fig. 2, bottom panel). In the absence of FDP Gi , a significantly enhanced consumption of NO is observed after addition of Rd in the presence of NADH and FlRd-red; however, if NO is readded to the solution, following the addition of FDP Gi , con-sumption of NO proceeds at a turnover rate of only ϳ0.2 s Ϫ1 . Moreover, this low rate of NO consumption was found to be essentially independent of the concentration of Rd. In conclusion, FDP Gi displays high O 2 -reductase activity, but very low NO-reductase activity, in agreement with the stopped-flow data described above.

The Three-dimensional Structure
The crystal structure of FDP Gi was solved at 1.9-Å resolution. The asymmetric unit contains two monomers forming a nonphysiological dimer that yields a tetramer by applying crystallographic symmetry. Such a tetramer consists of a dimer of dimers (Fig. 3A) and possibly represents the assembly of the protein in solution, as indicated by the HPLC data (see above). Upon formation of the physiological dimer ϳ10.9% of the ASA of each monomer becomes buried, whereas the ASA of each dimer decreases by ϳ12.9% in the tetrameric assembly. Interestingly, a similar homotetrameric arrangement has been reported for FDP Mm (16), but not for the homologous enzymes from D. gigas (6) and M. thermoacetica (14), whose structures were also solved; the latter two proteins were indeed reported to be dimers.
The two monomers in the asymmetric unit are completely superimposable (with a r.m.s.d. value calculated on the equivalent C␣ atoms of 0.13 Å). Each monomer is composed of two domains: a N-terminal ␤-lactamase-like domain containing the Fe-Fe center and a C-terminal flavodoxin-like domain containing the FMN cofactor (Fig. 3B): inspection of the Fo-Fc electron density maps reveals that the cofactor content is compatible with 0.8 -1.0 FMN and 1.9 -2.0 Fe per monomer. The ␤-lactamase-like domain (residues 4 -252 for chain A and 6 -252 for chain B) contains a sandwich of two ␤-sheets, each flanked on its outer face by three ␣-helices, and a two-stranded ␤-sheet that protrudes out from the central sandwich covering the Fe-Fe site. The flavodoxin-like domain (residues 253-412) contains five parallel ␤-strands, forming a central ␤-sheet surrounded on both sides by a total of five ␣-helices. Overall, the structure of FDP Gi and those available for bacterial flavodiiron proteins are similar (rmsd calculated on C␣ equivalent atoms of the dimers equal to 1.71, 1.7, and 1.84 Å for FDP Dg , FDP Mt , and FDP Mm , respectively).
Within each monomer the distance between the FMN and the Fe-Fe center is much too long (about 40 Å) to allow fast electron transfer between the two redox centers. In the physiological dimer, however, the monomers are in a head to tail arrangement that brings the Fe-Fe cluster of one monomer close to the FMN of the other one, thus allowing efficient electron transfer (Fig. 3A).
Three regions of the protein sequence account for most of the FMN contacts, namely the residues 265 SMYGTT 270 , 316 PTLNN 320 , 349 AFGWS 353 , and F382. The aromatic ring of Trp-352 is co-planar with the FMN isoalloxazine ring. This residue, almost conserved in FDPs, is likely involved in shuttling electrons between rubredoxin and FMN (16), and has been proposed (3) to account for the broadness of the 450 nm absorption band observed in most of the FPDs characterized so far. Additionally, the FMN moiety is contacted by a few residues of the nearby monomer (His-31, Glu-87, His-152, Trp-153, Ile-203, Leu-206, and Phe-207). His-31 is conserved in FDP Mt and Similarly to FDP Dg , FDP Mt , and FDP Mm , the active site of FPD Gi contains two irons (Fe1 and Fe2, the nearest to FMN) at a short distance (3.46 and 3.55 Å in each monomer) with an oxo (hydroxo or aquo) bridge, that is a common feature for di-iron proteins (39). Fe1 is ligated by the NE2 atoms of His-90 (2.14 Å) and His-230 (2.18 Å), by the OD2 atom of D89 (2.08 Å) and by the OD1 of Asp-171 (2.09 Å), the latter aspartate bridging the two iron atoms and coordinating Fe2 through the OD2 atom (2.08 Å). Fe2 is also coordinated by the NE2 of His-85 (2.29 Å), the NE2 of His-152 (2.14 Å) and the OE1 of Glu-87 (2.14 Å); the two latter residues are also involved in FMN binding. The oxo bridge is located at 1.94 and 1.97 Å from the Fe1 and Fe2, respectively. A cis peptide bond is present between Leu-151 and His-152, also detected in FDP Dg and in the active reduced form of FDP Mm (but not in FDP Mt ), and proposed to be necessary to project the imidazole ring of His-152 toward Fe1 (16). His-152 is located in a loop (Pro-149 -Pro-154) referred to as the switch loop in FDP Mm , where it undergoes a redox conformational change opening the binding site for the F 420 H 2 cofactor (16). Fig. 4A shows a superposition of the Fe-Fe site coordination sphere of FDP Mt , FDP Dg , and FDP Mm in the active form. All ligand residues occupy similar positions with the notable exception of the residue His-84 in FDP Dg (equivalent to His-90 in FDP Gi ). In FDP Dg this residue is stabilized in a non-bonding "out conformation" by interacting with an aspartate (Asp-225 in FDP Dg ), that is replaced by alanine or serine in all the other FPD structures solved to date, including FDP Gi ; consistently, in the latter enzymes this histidine residue coordinates the Fe atom. This structural difference was originally proposed (14) to account for the different specificity displayed by FDPs toward O 2 or NO. However, as discussed below, also based on our results, this hypothesis seems unlikely.
By inspection of the electron density map, a cloud of density is detected above the Fe-Fe center (Fig. S1 in supplemental data), compatible with an acetate or nitrate ion, both present in the crystallization medium. Such density was FIGURE 3. Overall structure of FDP Gi . A, tetrameric protein is shown as a transparent surface, with one of the functional dimers highlighted in ribbon representation (slate and magenta colors for the two monomers). FMN and the di-iron site depicted as yellow spheres. B, stereo view of one FDP Gi monomer in ribbon presentation with the N-terminal ␤-lactamase-like and the C-terminal flavodoxin-like domains colored in blue and red, respectively. FMN is represented in green sticks, the Fe atoms and the oxo (hydroxo or aquo) bridge as magenta and orange spheres, respectively. FEBRUARY 15, 2008 • VOLUME 283 • NUMBER 7 interpreted as a nitrate since this ion can make an additional H-bond with the His-31 NE2 atom (2.81 Å). Nitrate is bonded to both Fe atoms, thus occupying their sixth coordination position (Fig. 4B). Nitrate is located in the putative O 2 binding pocket in similar position where an O 2 molecule was found in FDP Dg and a H 2 O, an O 2 or an ethylene glycol molecule were found in the three structures of FDP Mt . In addition to the Fe ligating residues, the pocket is surrounded by five additional residues (Phe-30, His-31, His-176, Tyr-199, Ile-203) (Fig. 4B). Structural superposition of the putative O 2 binding pocket in FDPs (Fig. S2 in supplemental data) reveals that Tyr-199 is conserved and replaced by a phenylalanine only in FDP Mm ; Ile-203 is conserved or conservatively mutated, and His-176 is replaced in its topological position by an asparagine residue only in FDP Dg . Major differences are located above the pocket in the loop containing Phe-30 and His-31, that in FDP Gi is one residue shorter than in FDP Dg , two residues shorter than in FDP Mt and one residue longer than in FDP Mm . This difference allows His-31 to occupy the same position as His-25 in FDP Mt and His-26 in FDP Mm , whereas the same topological position is occupied by a tyrosine (Tyr-26) in FDP Dg . Interestingly, in FDP Mt mutation to phenylalanine of residues His-25 and Tyr-195 (corresponding to His-31 and Tyr-199 in FDP Gi , respectively) was shown to considerably lower the NO-reductase activity of this protein (14), presumably because involved in substrate binding.

DISCUSSION
G. intestinalis is an amitochondriate, microaerophilic parasite responsible for giardiasis, a common intestinal infectious disease and an important cause of morbidity in the developing countries. The disease is transmitted through fecaloral transfer of Giardia cysts, that after ingestion transform into trophozoites and colonize the small intestine; this causes various grades of symptoms, including nausea, stomach cramps, diarrhea, and vomiting, up to failure-to-thrive syndrome in children (23). Nitroimidazoles derivatives, particularly metronidazole, are widely used to treat the disease, though clinical resistance to these drugs has been repeatedly observed.
Giardia is a protozoon with an essentially glycolytic fermentative energy metabolism (25), leading to production of CO 2 , ethanol, alanine, and acetate. The parasite lacks the conventional respiratory enzymes, thus producing ATP by substrate level phosphorylation only (25). Relevant to this study, Giardia displays a significant sensitivity to O 2 (40), that was attributed to: (i) the expression of O 2 -labile key metabolic enzymes, such as pyruvate:ferredoxin oxidoreductase (PFOR, Ref. 41) and (ii) to the reactive oxygen species (ROS) produced by reaction of O 2 with NAD(P)H:menadione oxidoreductase (DT-diaphorase) (42). O 2 sensititivity is also enhanced by the fact that Giardia lacks the conventional ROS scavenging systems (26). Despite its O 2 sensitivity, in vivo the parasite is exposed to significant levels of O 2 in the luminal portion of duodenum and jejunum, where up to 50 M O 2 is present (43). Therefore, the occurrence of an efficient O 2 scavenging system is strictly required for survival and pathogenicity of Giardia.
Cells of the parasite were shown to be endowed with an O 2 consuming activity (40,44,45), that was attributed to a H 2 Oproducing FAD-containing NADH oxidase (27). This oxidase was thus proposed to be the enzyme responsible for protection of Giardia from O 2 .
We have investigated the structural ad functional properties of the FDP from G. intestinalis (FDP Gi ). This is one of the very few eukaryotic FPDs identified by genomic analyses, presumably acquired from prokaryotes by lateral gene transfer (19 -22,24). Our results clearly indicate that the enzyme displays high O 2 -reductase activity with formation of H 2 O (Ͼ40 s Ϫ1 ), but very low NO-reductase activity (ϳ0.2 s Ϫ1 ). The rate of O 2 reduction is so high that, even at the highest concentration of the reducing substrate tested ([Rd] ϭ 20 M), saturation is not achieved, thus preventing an estimate of V max ; of course, this may be partly due to limited efficiency of the nonphysiological reducing substrate used in the assays. In turnover with O 2 , FDP Gi does not display the significant inactivation reported for other FPDs (12,13), and O 2 is consumed following zero-order kinetics up to at least 10 M O 2 . Unlike NO, O 2 was found to react with the protein not only very rapidly, but also with high affinity (K m Յ 2 M, Figs. 1 and 2), yielding H 2 O as the product.
Based on the remarkable O 2 reactivity, we propose that FDP Gi plays a crucial role for in vivo O 2 detoxification. It remains to be established whether FDP Gi and NADH-oxidase work synergistically. In this respect, it is interesting to observe that sequence analysis of Giardia NADH-oxidase and FDP Gi suggested to us that these two proteins may belong to the same electron transport chain, accounting for O 2 detoxification. 6 FDP Gi does not seem to be involved in Giardia protection from nitrosative stress, as it is endowed with only very low NOreductase activity. NO is typically produced by the host immune system as part of the response to microbial infection (17,18). NO exerts cytostatic, but not cytotoxic effects toward Giardia (46), but the parasite counteracts the NO produced by nitric-oxide synthase (NOS) by actively consuming arginine (46). More recently, it has been proposed that Giardia infection might be cleared by the NO-induced stimulation of gastrointestinal motility (47), rather than by direct exposure of the pathogen to NO. Based on this information, it may not be surprising that FDP Gi was selected to scavenge O 2 much more efficiently than NO, unlike most of the bacterial FPDs characterized to date. A similar specificity toward O 2 was indeed reported only for the homologous protein from the methanogenic archaeon M. marburgensis (FDP Mm ) (16).
In the present study, the three-dimensional structure of the FDP from Giardia has been determined by crystallography (resolution 1.9 Å). The FDP Gi displays a tetrameric assembly consisting of a dimer of homodimers in a head-to-tail arrangement; a similar assembly was reported for the M. marburgensis enzyme (FDP Mm ) (16), where it was proposed to deal with thermoadaptation of this archaeal microorganism.
Overall, the structure of FDP Gi shows remarkable similarities with those of the few other prokaryotic members of the FDP family solved to date, namely the enzymes from D. gigas (FDP Dg (6)), M. thermoacetica (FDP Mt (14)), and M. marburgensis (FDP Mm (16)). A notable difference among the available structures is at the level of the residue His-90 that is a ligand of one of the two Fe atoms in the active site of all FDPs, except the one from D. gigas, where it is stabilized in a non-bonding "out" conformation. This difference was originally proposed (14) to account for the different specificity displayed by FDPs toward O 2 or NO: indeed, contrary to FDP Mt (13), the D. gigas enzyme consumes O 2 more efficiently than NO (7). However, the recently solved structure of FDP Mm (16) together with the one of the Giardia enzyme herein presented makes this hypothesis very unlikely. These two enzymes have a prevalent specificity for O 2 , being unable to efficiently metabolize NO; nonetheless, they display the above mentioned histidine residue His-90 liganded to Fe, like the enzyme from M. thermoacetica, which efficiently reduces NO to N 2 O (14).
More recently, Seedorf et al. (16) proposed that the ability of FDP Mm to process only O 2 (and not NO) could be attributed to two residues, Phe-198 and Tyr-25 (according to numbering of the FDP Mm enzyme), strictly conserved in FDPs from methanogenic Archaea. According to these authors Phe-198 is replaced by a Tyr and Tyr-25 by a Phe in those FPDs that are able to react not only with O 2 , but also with NO. Hence, the suggestion that Phe-198 and Tyr-25 may be crucial for the O 2 specificity. However, since in FDP Gi the latter residues are replaced by Tyr-199 and Phe-30 respectively, and this enzyme, like FDP Mm , displays a high reactivity with O 2 but no NO-reductase activity, this hypothesis may have to be abandoned, leaving open the issue of the O 2 versus NO specificity of FDPs.
It is also important to discover the physiological reducing substrate of the FPD enzyme in Giardia. According to the classification of FDPs proposed by Saraiva et al. (3), FDP Gi belongs to class A. Most of the FPDs of this class use rubredoxin as the reducing substrate, although in M. thermoacetica the latter protein is fused to a NAD(P)H:flavin oxidoreductase module in a single polypeptide chain, which was thus named "high molecular weight rubredoxin" (13). A notable exception is the archaeal enzyme from M. marburgensis, which accepts electrons from coenzyme F 420 , a 5-deazaflavin derivative present in relatively high concentrations in methanogenic archaea (15). As originally noticed by Seedorf et al. (16), in contrast to the F 420 -dependent enzymes, the FDPs using rubredoxin as the reducing substrate display a tryptophan residue stacked with the FMN cofactor, possibly involved in shuttling electrons between rubredoxin and FMN. Similarly to the other rubredoxin-dependent FDPs from D. gigas and M. thermoacetica, the latter residue (W352) is present and topologically conserved also in the Giardia enzyme. In analogy with FDP Dg and FDP Mt , Trp-352 is suitably solvent accessible and surrounded by several positively charged residues on the surface, that are likely involved in substrate recognition. Based on these structural similarities and on the ability of FDP Gi to be promptly reduced by the rubredoxin domain truncated from E. coli FlRd, rubredoxin seems a likely candidate substrate for FDP Gi , though as yet it remains to be detected in Giardia.
In conclusion, in the present study the first eukaryotic FDP from the human protozoan pathogen G. intestinalis proved to efficiently scavenge O 2 , thus appearing a good candidate to promote Giardia survival in the small intestine. The hypothesis, if validated by direct experiments on the parasites, might provide clues to alternative therapeutic strategies in the treatment of giardiasis.