The structural mechanism for iron uptake and release by transferrins

Transferrins are a group of iron-binding proteins that control the levels of iron in the body fluids of vertebrates by their ability to bind two Fe3+ and two CO3(2-). The transferrin molecule, with a molecular mass of about 80 kDa, is folded into two similarly sized homologous N- and C-lobes that are stabilized by many intrachain disulfides. As observed by X-ray crystallography, each lobe is further divided into two similarly sized domains, domain 1 and domain 2, and an Fe3+-binding site is within the interdomain cleft. Four of the six Fe3+ coordination sites are occupied by protein ligands (2 Tyr residues, 1 Asp, and 1 His) and the other two by a bidentate CO3(2-). Upon uptake and release of Fe3+, transferrins undergo a large-scale conformational change depending on a common structural mechanism: domains 1 and 2 rotate as rigid bodies around a rotation axis that passes through the two antiparallel beta-strands linking the domains. The extent of the rotation is, however, variable for different transferrin species and lobes. As a Fe3+ release mechanisms at low pH from the N-lobes of serum transferrin and ovotransferrin, the structural evidence for 'dilysine trigger mechanism' is shown. A structural mechanism for the Fe3+ release in presence of a non-synergistic anion is proposed on the basis of the sulfate-bound apo crystal structure of the ovotransferrin N-lobe. Domain-opened structures with the coordinated Fe3+ by the two tyrosine residues are demonstrated in fragment and intact forms, and their functional implications as a possible intermediate for iron uptake and release are discussed.     

Differences in the protein fluorecence of the two iron(III)-binding sites of ovotransferrin.

1. Changes in the tryptophan fluorescence and the visible absorption spectrum resulting from the combination of apo-ovotransferrin with Fe3+, F,E2+, Cu2+, Zn2+, Mn2+, and Cd2+were measured. 2. As expected for a radiationless transfer of electronic excitation energy, only the ions Fe3+, Fe2+and Cu2+, which gave complexes with large extinctions between 300 and 370nm, resulted in large decreases in trytophan fluorescence. 3. The decrease in protein fluorescence was non-linear with increasing occupancy of the Fe3+ -and Cu2+ - binding sites. The decrease in fluorescence on binding of Fe3+ was biphasic and showed that the two metal-binding sites were being occupied sequentially at pH7.4-8.4. The first site reacted with Fe3+ instantaneously, the second was occupied over a minute. 5. The nonidentity of the two sites was also demonstrated by the preparation of a stable hybrid containing both Cu2+ and Zn2+.h Cu2+ and Zn2+     

CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers

DNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. The data show a single 5- or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30× more stable than Dps1; and the lower limit of K(D) for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.     

Kinetics of the release of aluminum from human serum dialuminum transferrin to citrate

The kinetics of release of Al3+ from human serum dialuminum transferrin (Al2Tf) to citrate were investigated at 37 degrees C, pH 7.4, mu = 0.7 M, by difference UV spectrophotometry. The two metal-binding sites are not identical but behave in a kinetically similar manner to give apparent second-order rate constants of 0.60 and 0.38 M-1 s-1, respectively, for release of the first Al3+ from Al2Tf. The rate constants for release of the second metal ion from the monoaluminum transferrins are 0.27 and 0.12 M-1 s-1. The kinetic scheme for release of A13+ from Al2Tf is therefore similar to that for release of Fe3+ from Fe2Tf, but the rate of constants for metal ion release are between two and four orders of magnitude larger.     

Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.     

The iron-binding properties of hen ovotransferrin.

1. The distribution of iron between the two iron-binding sites in partially saturated ovotransferrin was studied by labelling with 55Fe and 59Fe and by gel electrophoresis in a urea-containing buffer. 2. When iron is added in the form of chelate complexes at alkaline pH, binding occurs preferentially at the N-terminal binding site. In acid, binding occurs preferentially at the C-terminal site. 3. When simple iron donors (ferric and ferrous salts) are used the metal is distributed at random between the binding sites, as judged by the gel-electrophoresis method. The double-isotope method shows a preference of ferrous salts for the N-terminal site. 4. Quantitative treatment of the results of double-isotope labelling suggests that in the binding of iron to ovotransferrin at alkaline pH co-operative interactions between the sites occur. These interactions are apparently absent in the displacement of copper and in the binding of iron at acid pH.     

The displacement of copper by iron at the specific binding sites of ovotransferrin

We have examined the kinetics and mechanism by which iron can displace copper at the specific metal-binding sites of ovotransferrin. Fe2+ was added to Cu2+-ovotransferrin-CO3(2-) in the presence of NaHCO3 and ambient O2. The reaction has been followed by standard and stopped-flow spectrophotometry, EPR spectroscopy and analysis of chromogen-reactive Fe2+. The reaction is best described as triphasic. An initial jump in absorbance takes place in the first 2 s. In the next minute there is a further increase in absorbance and shift in the spectral maximum from 440 to 446 nm. The third phase is complex. The bulk of the spectrophotometric change, a decrease in absorbance with a shift to a maximum of 453 nm, lasts approx. 3 min. Minor spectral and EPR changes, however, take place over the next several hours. Chromogenic analysis of Fe2+ indicates that approx. 1 min is required to oxidize the Fe2+. EPR spectra reveal the formation of an Fe3+-ovotransferrin complex within the first 20 s; however, this lacks the characteristic doublet of specific Fe3+-ovotransferrin-CO3(2-). The simultaneous presence of specific Cu2+-ovotransferrin-CO3(2-) and Fe3+-ovotransferrin-CO3(2-) signals suggests a period in which the protein specifically binds both metal ions perhaps resulting from a differential reactivity of the two metal-binding sites. The addition of Cu(NO3)2 to Fe3+-ovotransferrin-CO3(2-) resulted in a complex with specific Fe3+ and non-specific Cu2+. The EPR spectrum of this complex and the final product of our displacement reaction were virtually identical. Distinct parallels in reaction of Cu2+-ovotransferrin-CO3(2-) with Fe(NH4)2(SO4)2, Fe(NO3)3 and Fe3+-nitrilotriacetic acid were observed. A reaction sequence involving the binding and oxidation of non-specific Fe2+ followed by Cu2+ displacement by Fe3+ at the specific sites and binding of non-specific Cu2+ is suggested.     

Redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the gav = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983.(ABSTRACT TRUNCATED AT 250 WORDS)     

A kinetic study of the endogenous reduction of the oxidized sites in the primary cytochrome c peroxidase-hydrogen peroxide compound.

The primatry compound formed in the reaction between H2O2 and cytochrome c peroxidase is oxidized two equivalents above the native enzyme. The two oxidized sites are thought to be an Fe(IV) and an amino acid radical. In the absence of oxidizable substrate, the Fe(IV) and radical sites decay by apparent first-order processes but at different rates. It is likely that the decay involves both intra- and intermolecular electron-transfer reactions. The reduction of the Fe(IV) site depends upon the pH with a minimum reduction rate of 2.9-10(-5)s(-1) at pH 6. At pH 4 and 6, the reduction of the Fe(IV) site is facilitated by prior oxidation of amino acid residues in the protein.     

Bacterial diversity in Fe‐rich hydrothermal sediments at two South Tonga Arc submarine volcanoes

Seafloor iron oxide deposits are a common feature of submarine hydrothermal systems. Morphological study of these deposits has led investigators to suggest a microbiological role in their formation, through the oxidation of reduced Fe in hydrothermal fluids. Fe‐oxidizing bacteria, including the recently described Zetaproteobacteria, have been isolated from a few of these deposits but generally little is known about the microbial diversity associated with this habitat. In this study, we characterized bacterial diversity in two Fe oxide samples collected on the seafloor of Volcanoes 1 and 19 on the South Tonga Arc. We were particularly interested in confirming the presence of Zetaproteobacteria at these two sites and in documenting the diversity of groups other than Fe oxidizers. Our results (small subunit rRNA gene sequence data) showed a surprisingly high bacterial diversity, with 150 operational taxonomic units belonging to 19 distinct taxonomic groups. Both samples were dominated by Zetaproteobacteria Fe oxidizers. This group was most abundant at Volcano 1, where sediments were richer in Fe and contained more crystalline forms of Fe oxides. Other groups of bacteria found at these two sites include known S‐ and a few N‐metabolizing bacteria, all ubiquitous in marine environments. The low similarity of our clones with the GenBank database suggests that new species and perhaps new families were recovered. The results of this study suggest that Fe‐rich hydrothermal sediments, while dominated by Fe oxidizers, can be exploited by a variety of autotrophic and heterotrophic micro‐organisms.     

Mössbauer studies of electrophoretically purified monoferric and diferric human transferrin

Summary Electrophoretically purified⁵⁷Fe-enriched monoferric and diferric human transferrins and selectively labeled complexes ([C-⁵⁶Fe,N-⁵⁷Fe]transferrin and [C-⁵⁷Fe,N-⁵⁶Fe]transferrin) were studied by Mössbauer spectroscopy. The data were recorded at 4.2 K over a wide range of applied magnetic fields (0.05–6 T) and were analyzed by a spin-Hamiltonian formalism. Characteristic hyperfine parameters were found and the obtained zero-field splitting parameters (D=0.25±0.05 cm^–1 andE/D = 0.30 ± 0.02) agree with previous electron paramagnetic resonance (EPR) findings. The weak-field spectra of the [N-⁵⁷Fe]transferrin are slightly broader than those of the [C-⁵⁷Fe]transferrin, indicating that the N-terminal iron site may be more heterogeneous. However, the absorption line positions and the relative intensities of the subspectra originating from the three Kramers doublets of each Fe³⁺ site are identical. Thus the electronic structures of the two iron sites can be described by the same set of spin- Hamiltonian parameters, indicating that the ligand environments for the two sites are the same, as suggested by the recent X-ray crystallographic studies. This suggestion is further supported by the observation that the strong-field spectra of the two monoferric transferrins are indistinguishable. The selectively labeled mixed-isotope transferrins exhibit spectra that are identical to those of the corresponding monoferric⁵⁷Fe-enriched transferrins, implying that the occupation of one iron site has little or no effect on the immediate envirnoment of the other site, a finding that is not surprising since the two sites are separated by approximately 4.2 nm.     

A possible role for the conserved trimer interface of ferritin in iron incorporation.

Ferritin is a large protein, highly conserved among higher eukaryotes, which reversibly stores iron as a mineral of hydrated ferric oxide. Twenty-four polypeptides assemble to form a hollow coat with the mineral inside. Multiple steps occur in iron core formation. First, Fe2+ enters the protein. Then, several alternate paths may be followed which include oxidation at site(s) on the protein, oxidation on the core surface, and mineralization. Sequence variations occur among ferritin subunits which are classified as H or L; Fe2+ oxidation at sites on the protein appears to be H-subunit-specific or protein-specific. Other steps of ferritin core formation are likely to involve conserved sites in ferritins. Since incorporation of Fe2+ into the protein must precede any of the other steps in core formation, it may involve sites conserved among the various ferritin proteins. In this study, accessibility of Fe2+ to 1,10-phenanthroline, previously shown to be inaccessible to Fe2+ inside ferritin, was used to measure Fe2+ incorporation in two different ferritins under various conditions. Horse spleen ferritin (L/H = 10-20:1) and sheep spleen ferritin (L/H = 1:1.6) were compared. The results showed that iron incorporation measured as inaccessibility of Fe2+ to 1,10-phenanthroline increased with pH. The effect was the same for both proteins, indicating that a step in iron core formation common among ferritins was being measured. Conserved sites previously proposed for different steps in ferritin core formation are at the interfaces of pairs and trios of subunits. Dinitrophenol cross-links, which modify pairs of subunits and affect iron oxidation, had no effect on Fe2+ incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron-Stress Response in Tomato (Lycopersicon esculentum) 1. Sites of Fe Reduction, Absorption and Transport

T3238fer (Fe-inefficient) and T3238FER (Fe-efficient) tomato plants differ in their ability to utilize Fe and therefore can be used as test genotypes to locate sites of Fe uptake or to characterize changes that occur in roots in response to Fe stress (Fe deficiency). T3238fer does not respond to Fe stress. Release of hydrogen ions and reduction of Fe³⁺ to Fe²⁺ are two primary responses of T3238FER roots to Fe stress. Fe reduction sites were predominately in the young lateral roots, and between the regions of root elongation and maturation of the primary root. The use of BDPS (bathophenanthrolinedisulfonate) to trap Fe²⁺ did not affect the release of H⁺ ions or reduction by T3238FER roots. BPDS did not decrease Fe uptake until it exceeded the Fe concentration in the nutrient solution. A sevenfold increase in BPDS caused a threefold decrease in Fe taken up by the plant. Fe³⁺ is reduced to Fe²⁺ at root sites accessible to BPDS. Adding Zn decreased the response to Fe stress. Iron stress initiates the development of lateral roots, and we propose that most Fe enters the plant through these roots. The iron moves through protoxylem into the metaxylem of the primary root and then to the top of the plant as Fe citrate. Root environmental factors that are competitive or inhibit Fe-stress response, or genotypes that fail to respond to Fe stress, contribute to the development of Fe deficiency in plants.     

Superoxide dismutases: active sites that save, but a protein that kills

Protection from oxidative damage is sufficiently important that biology has evolved three independent enzymes for hastening superoxide dismutation: the Cu- and Zn-containing superoxide dismutases (Cu,Zn-SODs), the SODs that are specific for Fe or Mn or function with either of the two (Fe-SODs, Mn-SODs or Fe/Mn-SODs), and the SODs that use Ni (Ni-SODs). Despite the overwhelming similarities between the active sites of Fe-SOD and Mn-SOD, the mechanisms and redox tuning of these two sites appear to incorporate crucial differences consistent with the differences between Fe3+/2+ and Mn3+/2+. Ni-SOD is revealed by spectroscopy to employ completely different ligation to that of the other SODs while nonetheless incorporating a device also found in Cu,Zn-SOD. Finally, the protein of human Cu,Zn-SOD appears to be an important contributor to the development of amyotrophic lateral sclerosis, possibly because of its propensity for extended beta-sheet formation.     

EPR detection of protein-derived radicals in the reaction of H(2)O(2) with Fe bound in mitochondrial F(1)ATPase.

A severe inactivation is obtained upon the addition of H(2)O(2) to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H(2)O(2) produces the complete oxidation of Fe(II) to Fe(III) and the concomitant appearance of two protein-derived radical species. The two signals (g = 2.036 and g = 2.007) display a different temperature dependence and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers. Such events are not observed when H(2)O(2) is added either to native F(1)ATPase containing a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(III) and Fe(II), intramolecular long-range electron transfer may occur from Fe(II) to a high oxidation state species of Fe formed in the nucleotide-independent site upon oxidation of Fe(III) by H(2)O(2).     

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] foot-printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.     

Ferrous ion binding to recombinant human H-chain ferritin. An isothermal titration calorimetry study

Iron deposition within the iron storage protein ferritin involves a complex series of events consisting of Fe(2+) binding, transport, and oxidation at ferroxidase sites and mineralization of a hydrous ferric oxide core, the storage form of iron. In the present study, we have examined the thermodynamic properties of Fe(2+) binding to recombinant human H-chain apoferritin (HuHF) by isothermal titration calorimetry (ITC) in order to determine the location of the primary ferrous ion binding sites on the protein and the principal pathways by which the Fe(2+) travels to the dinuclear ferroxidase center prior to its oxidation to Fe(3+). Calorimetric titrations show that the ferroxidase center is the principal locus for Fe(2+) binding with weaker binding sites elsewhere on the protein and that one site of the ferroxidase center, likely the His65 containing A-site, preferentially binds Fe(2+). That only one site of the ferroxidase center is occupied by Fe(2+) implies that Fe(2+) oxidation to form diFe(III) species might occur in a stepwise fashion. In dilute anaerobic protein solution (3-5 microM), only 12 Fe(2+)/protein bind at pH 6.51 increasing to 24 Fe(2+)/protein at pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding of Fe(2+) to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe(2+) binding. The total Fe(2+) binding capacity of the protein is reduced in the 3-fold hydrophilic channel variant S14 (D131I+E134F), indicating that the primary avenue by which Fe(2+) gains access to the interior of ferritin is through these eight channels. The binding stoichiometry of the channel variant is one-third that of the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe(2+) binding are similar for the two with an average values (DeltaH degrees = 7.82 kJ/mol, binding constant K = 1.48 x 10(5) M(-)(1) at pH 7.04). Since channel mutations do not completely prevent Fe(2+) binding to the ferroxidase center, iron gains access to the center in approximately one-third of the channel variant molecules by other pathways.     

Binding of transferrin-iron to the plasma membrane of a lactating rabbit mammary gland cell

1. Cells isolated from a lactating rabbit mammary gland have been investigated for transferrin-iron receptors. The existence of these structures has been demonstrated through a specific binding with a competitor non-labelled rabbit transferrin. 2. The interaction of iron-receptor complex depends on the concentration of [59Fe]transferrin, the number of cells and the time. 3. Scatchard's plot of data indicates two classes of receptor sites: one with a binding capacity 6.48 x 10(-9) g Fe per cell and affinity constant 2.48 x 10(10) M-1 and another with 1.06 x 10(-8) g Fe per cell and 4.66 x 10(11) M-1 respectively. 4. The probable mechanism of the iron transport from blood to milk through the lactating cell was discussed.     

Proximity of transmembrane segments M3 and M1 of the alpha subunit of Na+,K+-ATPase revealed by specific oxidative cleavage mediated by a complex of Cu2+ ions and 4,7-diphenyl-1,10-phenanthroline

This paper describes a novel approach to specific oxidative cleavage of Na(+),K(+)-ATPase, mediated by Cu(2+) ions and a hydrophobic phenanthroline, 4,7-diphenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H(2)O(2). The cleavage produces two major fragments of the alpha subunit, with apparent molecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entrance of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu(2+)-DPP complex. We infer that M3 and M1 are in proximity near the cytoplasmic surface. The yields of 96.5 and 76 kDa fragments are not significantly affected by ligands that stabilize different E(1) and E(2) conformations. In E(2)(K) and E(2)P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 96.5 and 76 kDa fragments are indistinguishable from two fragments near M3 and M1 produced by Fe(2+)-catalyzed cleavage described previously [Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], whereas other Fe(2+)-catalyzed cleavage fragments in the cytoplasmic P and A domains are not observed with the Cu(2+)-DPP complex. These findings provide experimental support for the concept of two separate Fe(2+) sites. A homology model, with Na(+),K(+)-ATPase residues within transmembrane segments and connecting loops substituted into the crystal structure of Ca(2+)-ATPase, shows the proximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasmic surface. Thus, the model strongly supports the conclusions based on cleavages mediated by the Cu(2+)-DPP complex (or Fe(2+) at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na(+),K(+)-ATPase and Ca(2+)-ATPase.     

Formation of monoferric ovotransferrins in the presence of chelates.

1. When ovotransferrin is partially saturated with iron, endotherms for apo-ovotransferrin, two monoferric ovotransferrins and Fe2-ovotransferrin are observed by differential scanning calorimetry. The relative sizes of the endotherms are changed in the presence of the iron-chelating agents nitrilotriacetic acid and ATP. 2. When iron is added as Fe(III)-nitrilotriacetate, at Fe-nitrilotriacetate: ovotransferrin ratios less than unity, the endotherm for Fe2-ovotransferrin is essentially absent. At Fe-nitrilotriacetate: ovotransferrin ratios of unity the only species present in solution in appreciable concentration as evidenced by their differential-scanning-calorimetry endotherms, are two monoferric ovotransferrins in approximately equal amounts. At Fe-nitrilotriacetate: ovotransferrin ratios greater than unity, the apo-ovotransferrin endotherm is absent, and the endotherms for the two monoferric ovotransferrins decrease in size as the endotherm for Fe2-ovotransferrin increases. 3. In the presence of nitrilotriacetate, binding of iron to the two sites of ovotransferrin is highly anti-co-operative, but essentially indiscriminate. When monoferric ovotransferrin is formed from apo-ovotransferrin, binding at one site is slightly favoured compared with binding at the other site, but once iron has been bound at either site, the binding affinity for iron at the unoccupied site is much decreased.