Effects of iron deficiency on iron binding and internalization into acidic vacuoles in Dunaliella salina

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe(3+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.     

Crystallographic and biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein.

The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe(3+)-binding protein (hFbp) has been determined to 1.75 A resolution. Information from this structure complements that derived from the holo structure with respect to the delineation of the process of iron binding and release. A 21 degrees rotation separates the two structural domains when the apo form is compared with the holo conformer, indicating that upon release of iron, the protein undergoes a change in conformation by bending about the central beta-sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary binding site anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphate with an affinity K(d) of 2.3 x 10(-3) M. The presence of this ternary binding site anion appears to arrange the C-terminal iron-binding residues conducive to complementary binding to Fe(3+), while residues in the N-terminal binding domain must undergo induced fit to accommodate the Fe(3+) ligand. These observations suggest a binding process, the first step of which is the binding of a synergistic anion such as phosphate to the C-terminal domain. Next, iron binds to the preordered half-site on the C-terminal domain. Finally, the presence of iron organizes the N-terminal half-site and closes the interdomain hinge. The use of the synergistic anion and this iron binding process results in an extremely high affinity of the Fe(3+)-binding proteins for Fe(3+) (nFbp K'(eff) = 2.4 x 10(18) M(-1)). This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe(3+)-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.     

Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants

Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).     

Mutational analysis of the iron binding site of Saccharomyces cerevisiae ferroxidase Fet3. An in vivo study.

The role of residues predicted to be involved in the binding of iron by the yeast ferroxidase Fet3 has been studied by site-directed mutagenesis. The effect of Fet3 mutations E185A, E185Q, Y354F, D409V and H489D has been investigated in vivo by kinetic analyses of high affinity iron uptake. Our results indicate that Glu-185 is critical for the binding of iron, since substitution of this residue with Ala or Gln strongly affects both growth and the kinetic parameters of high affinity iron uptake, greatly increasing K(m). Mutations Y354F and D409V result in less severe alteration of high affinity iron uptake, while mutant H489D is unable to grow under conditions of iron limitation.     

Discovery of native metal ion sites located on the ferredoxin docking side of photosystem I

Photosystem I (PSI) is a large membrane protein that catalyzes light-driven electron transfer across the thylakoid membrane from plastocyanin located in the lumen to ferredoxin in the stroma. Metal analysis reveals that PSI isolated from the cyanobacterial membranes of Synechococcus leopoliensishas a near-stoichiometric 1 molar equiv of Zn (2+) per PSI monomer and two additional surface metal ion sites that favor Cu (2+) binding. Two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy reveals coupling to the so-called remote nitrogen of a single histidine coordinated to one of the Cu (2+) centers. EPR and X-ray absorption fine structure (XAFS) studies of 2Cu-PSI complexes reveal the direct interaction of ferredoxin with the Cu (2+) centers on PSI, establishing the location of native metal sites on the ferredoxin docking side of PSI. On the basis of these spectroscopic results and previously reported site-directed mutagenesis studies, inspection of the PSI crystal structure reveals a cluster of three highly conserved residues, His(D95), Glu(D103), and Asp(C23), as a likely Cu (2+) binding site. The discovery of surface metal sites on the acceptor side of PSI provides a unique opportunity to probe the stromal region of PSI and the interactions of PSI with its reaction partner, the soluble electron carrier protein ferredoxin.     

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.     

Kinetic and mutational analyses of the major cytosolic exopolyphosphatase from Saccharomyces cerevisiae

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg(2+) (K(d) of 24 microm) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg(2+) in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK(a) of 4.6 and 5.6) is modulated by ionization of a third group (pK(a) of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg(2+) affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co(2+) bound to the weak binding site versus the Mg(2+)- or Mn(2+)-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase.     

C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.     

Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.     

Directed cleavage of RNA with protein-tethered EDTA-Fe

There are several methods for locating the RNA site where a protein binds. One of the less common methods is directed cleavage of the RNA by an EDTA-Fe reagent tethered to the protein. The reaction of the EDTA-Fe(III) with ascorbate or hydrogen peroxide produces reactive oxygen species, such as hydroxyl radicals, localized within a 10-A radius of the iron center. The reactive oxygen species will attack the ribose or deoxyribose of nucleic acids as well as proximal polypeptide backbones. One EDTA-Fe reagent, (EDTA-2-aminoethyl)-2-pyridyl disulfide complexed to iron (EPD-Fe), has been tethered to several proteins through a disulfide linkage to engineered cysteine thiols and used to cleave DNA, proteins, and RNA. A second tethered EDTA-Fe reagent, 1-(p-bromoacetamidobenzyl)-EDTA-Fe, or BABE, has also been used to cleave RNA. Here we describe the issues involved in using these reagents with any RNA binding protein.     

Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes

Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.     

Iron binding to horse spleen apoferritin: a vanadyl ENDOR spin probe study.

The role of the protein shell in the formation of the hydrous ferric oxide core of ferritin is poorly understood. A VO2+ spin probe study was undertaken to characterize the initial complex of Fe2+ with horse spleen apoferritin (96% L-subunits). A competitive binding study of VO2+ and Fe2+ showed that the two metals compete 1:1 for binding at the same site or region of the protein. Curve fitting of the binding data showed that the affinity of VO2+ for the protein was 15 times that of Fe2+. Electron nuclear double resonance (ENDOR) measurements on the VO(2+)-apoferritin complex showed couplings from two nitrogen nuclei, tentatively ascribed to the N1 and N3 nitrogens of the imidazole ligand of histidine. The possibility that the observed nitrogen couplings are from two different ligands is not precluded by the data, however. A pair of exchangeable proton lines with a coupling of approximately 1 MHz is tentatively assigned to the NH proton of the coordinated nitrogen. A 30-40% reduction in the intensity of the 1H matrix ENDOR line upon D2O-H2O exchange indicates that the metal-binding site is accessible to solvent and, therefore, to molecular oxygen as well. The ENDOR data provide the first evidence for a principle iron(II)-binding site with nitrogen coordination in an L-subunit ferritin. The site may be important in Fe2+ oxidation during the beginning stages of core formation.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

Kinetic characterization of the interaction between cytochrome oxidase and cytochrome c

The mechanism of electron transfer catalyzed by cytochrome oxidase was investigated by monitoring the reaction of cytochrome oxidase with cytochrome c under carefully controlled anaerobic conditions. The kinetics of the reaction were examined by varying conditions of ionic strength, inhibitor binding, and oxidation-reduction potential. An analogue of cytochrome c in which the iron atom was replaced with cobalt was used to probe the effect of redox potential on the reaction. Under conditions of low ionic strength, there is very rapid oxidation of cytochrome c and reduction of oxidase which occurs at a rate of 3 X 10(7) M-1 s-1. The number of electrons transferred exhibit a hyperbolic dependence on the concentration of cytochrome c reaching a maximum of 2 electrons transferred at the highest concentration of reduced cytochrome c employed. The total number of electrons transferred was always observed to be distributed equally between cytochrome a and a second acceptor which appears to be the associated copper center; electron transfer to cytochrome a3 did not occur in the absence of oxygen. Substitution of cytochrome c by the cobalt analogue (which represents a decrease in oxidation-reduction potential of about 400 mV) yielded identical results indicating that the origin of the lack of reactivity of cytochrome a3 is of a kinetic nature. The effect of increasing the ionic strength on the reaction was 2-fold: a marked decrease in reaction rate and the appearance of biphasic kinetics with the amplitude of the very fast absorbance changes at 605 nm decreasing from 80% to 40% of the total anticipated from static absorbance measurements. Each of the two phases accounted for a maximum of 1 electron at the highest ionic strength employed. These results are simulated in terms of a sample kinetic reaction scheme involving a two-step electron transfer at one binding site.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.     

Cooperative setting for long-range linkage of Ca(2+) binding and ATP synthesis in the Ca(2+) ATPase

High-affinity and cooperative binding of two Ca(2+) per ATPase (SERCA) occurs within the membrane-bound region of the enzyme. Direct measurements of binding at various Ca(2+) concentrations demonstrate that site-directed mutations within this region interfere selectively with Ca(2+) occupancy of either one or both binding sites and with the cooperative character of the binding isotherms. A transition associated with high affinity and cooperative binding of the second Ca(2+) and the engagement of N796 and E309 are both required to form a phosphoenzyme intermediate with ATP in the forward direction of the cycle and also to form ATP from phosphoenzyme intermediate and ADP in the reverse direction of the cycle. This transition, defined by equilibrium and kinetic characterization of the partial reactions of the enzyme cycle, extends from transmembrane helices to the catalytic site through a long-range linkage and is the mechanistic device for interconversion of binding and phosphorylation potentials.     

An apparently allosteric effect involving N2O with the nitrous oxide reductase from Wolinella succinogenes.

It was shown that kcat for the benzyl viologen cation (BV+)-N2O oxidoreductase activity of nitrous oxide reductase from Wolinella succinogenes was 2-3 times greater at high N2O concentrations than at low. This effect of N2O on kcat exhibited a titration curve implicating a single secondary binding site for N2O with a Kd of 130-200 microM (Km with respect to N2O is about 2.5 microM). This work represents the first evidence of an apparently allosteric kinetic effect among nitrous oxide reductases. Its possible cause is discussed. BV+ was generated in these kinetic studies by addition of sub-stoichiometric amounts of dithionite. This means of reduction proved to be superior to the photochemical generation of BV+ that had been used previously with the enzyme. Mass spectrometric measurements suggested that the M(r) of the subunit of the enzyme is about 95,500 rather than 88,000.     

Protein intermediate trapped by the simultaneous crystallization process. Crystal structure of an iron-saturated intermediate in the Fe3+ binding pathway of camel lactoferrin at 2.7 a resolution

This is the first protein intermediate obtained in the crystalline state by the simultaneous process of Fe(3+) binding and crystal nucleation and is also the first structure of an intermediate of lactoferrin in the Fe(3+) binding pathway. Lactoferrin is an iron-binding 80-kDa glycoprotein. It binds Fe(3+) very tightly in a closed interdomain cleft in both lobes. The iron-free structure of lactoferrin, on the other hand, adopts an open conformation with domains moving widely apart. These studies imply that initial Fe(3+) binding must be in the open form. The protein intermediate was crystallized by the microdialysis method. The protein solution, with a concentration of 100 mg/ml in 10 mm Tris-HCl, pH 8.0, was loaded in a capillary and dialyzed against the same buffer containing 26% (v/v) ethanol placed in a reservoir. FeCl(3) and CO(3)(2-) in excess molar ratios to that of protein in its solution were added to the reservoir buffer. The crystals appeared after some hours and grew to the optimum size within 36 h. The structure was determined by molecular replacement method and refined to final R- and R-free factors of 0.187 and 0.255, respectively. The present structure showed that the protein molecule adopts an open conformation similar to that of camel apolactoferrin. The electron density map clearly indicated the presence of two iron atoms, one in each lobe with 4-fold coordinations: two by the protein ligands of Tyr-92(433) OH and Tyr-192(526) OH and two other coordination sites occupied by oxygen atoms of bidentate CO(3)(2-) ions leading to a tetrahedral intermediate. The CO(3)(2-) anion is stabilized through hydrogen bonds with the synergistic anion-binding site Arg-121(463) and with Ser-122 Ogamma in the N-lobe and Thr-464 Ogamma in C-lobe. The third oxygen atom of CO(3)(2-) interacts with a water molecule in both lobes.     

Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage.

Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I.