Selective adsorption of apoferritin on immobilized Fe(III): demonstration of Fe(III) binding sites

Immobilized metal ion affinity chromatography has been used to demonstrate and partially characterize Fe(III) binding sites on apoferritin. Binding of Fe(III) to these sites is influenced by pH, but not affected by high ionic strength. These results suggest that both ionic and coordinate covalent interactions are important in the formation of the Fe(III): apoferritin complex. This is, to our knowledge, the first demonstration of direct Fe(III) binding to apoferritin. Other immobilized metal ions, including Zn(II), Ni(II), Cu(II), Cr(III), Co(II), and Tb(III), displayed little or no adsorption of apoferritin. The analytical technique of immobilized metal ion affinity chromatography also shows great promise in the purification of apoferritin, ferritin, and other iron-binding proteins.     

Binding of terbium to apoferritin: a fluorescence study

Luminescence measurements show that apoferritin binds three Tb(III) atoms per subunit in accordance with crystallographic evidence. Fe(II) competes with Tb(III) for at least some of the binding sites. This competition may be the molecular basis for the inhibition of iron incorporation into apoferritin brought about by Tb(III). Ca(II), which is generally replaced by Tb(III) in Ca(II) binding proteins, does not compete with the lanthanide for binding to apoferritin.     

Competition studies in horse spleen ferritin probed by a kinetically inert inhibitor, [Cr(TREN)(H2O)(OH)]2+, and a highly luminescent Tb(III) reagent

The ability of ferritin as an Fe(II) detoxifier and Fe(III) storage protein is limited by its ability to recognize and incorporate Fe(II), which is then oxidized and mineralized at internal protein sites. The Cr(III) amine complex [Cr(N(CH(2)CH(2)NH(2))(3)(H(2)O)(OH)](2+) [abbreviated as Cr(TREN)] is a kinetically inert inhibitor of iron incorporation and mineralization in ferritin. Unlike other inhibitors, Cr(TREN) can only exchange its two aqua/hydroxy ligands. Competition studies between Cr(TREN) and Tb(III) binding have been performed in horse spleen ferritin (HoSF) to probe uptake of Fe(II). From these studies, we propose that Cr(TREN) inhibits Fe(II) uptake by obstructing the routes of metal uptake and by disrupting the early recognition events at the protein surface that precede metal ion uptake. Using an improved luminescence approach to quantify Tb(III) binding to the protein, we demonstrate that Tb(III) cannot interfere with Cr(TREN) binding to ferritin, but that Cr(TREN) dramatically inhibits Tb(III) binding. We show that bound Tb(III) serves as a reliable reporter for Cr(TREN) binding, as the latter efficiently quenches the Tb(III) luminescence via inter-ion energy transfer. Two types of Cr(TREN) binding sites were successfully distinguished from these competition experiments. A common Tb(III)/Cr(TREN) site was identified with stoichiometry of approximately 0.6 equivalents of metal cation per ferritin subunit. We propose that the sites along the three-fold channels and the ferroxidase sites are common binding sites for Tb(III) and Cr(TREN). The remaining Cr(TREN) (2.4 equivalents of metal ions/subunit) does not compete with Tb(III) but rather blocks Tb(III) access into the cavity and decreases the protein's affinity for Tb(III).     

Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies

The knowledge of the route through which iron can enter and leave the apoferritin shell is a prerequisite for the understanding of ferritin's function. The involvement of the hydrophilic 3-fold channels in the iron uptake process has been studied by taking advantage of the reactivity of specific residues that line such channels, i.e., glutamic acid-127 and aspartic acid-130, the major Cd(II) binding sites, and cysteine-126. 113Cd NMR experiments have provided direct evidence for the competition between Fe(II) and Cd(II) binding to major Cd(II) binding sites on the protein and or a higher affinity of Fe(II) for these sites, in line with the well-known inhibitory effect of Cd(II) on iron uptake. Further evidence for the use of the 3-fold channels in the iron entry process has been obtained by means of chemical modification of Cys-126 with different mercurials. In particular, the introduction of the additional carboxylate carried by p-(chloromercuri)benzoate near Asp-127 and Glu-130 increases the initial rate of iron uptake and affects the coordination geometry of the metal in the Fe(III)-apoferritin complex as indicated by optical absorption and EPR data. The assignment of these effects to the carboxylate moiety of p-(chloromercuri)benzoate is brought out by the observation that the introduction in the 3-fold channel of the benzene ring only by means of phenylmercuric acetate has no effect on the initial iron uptake kinetics and on the spectroscopic properties of the Fe(III)-apoferritin complex.     

Defining metal ion inhibitor interactions with recombinant human H- and L-chain ferritins and site-directed variants: an isothermal titration calorimetry study

Zinc and terbium, inhibitors of iron incorporation in the ferritins, have been used for many years as probes of structure-function relationships in these proteins. Isothermal titration calorimetric and kinetic measurements of Zn(II) and Tb(III) binding and inhibition of Fe(II) oxidation were used to identify and characterize thermodynamically ( n, K, Delta H degrees, Delta S degrees, and Delta G degrees ) the functionally important binding sites for these metal ions in recombinant human H-chain, L-chain, and H-chain site-directed variant ferritins. The data reveal at least two classes of binding sites for both Zn(II) and Tb(III) in human H-chain ferritin: one strong, corresponding to binding of one metal ion in each of the eight three-fold channels, and the other weak, involving binding at the ferroxidase and nucleation sites of the protein as well as at other weak unidentified binding sites. Zn(II) and Tb(III) binding to recombinant L-chain ferritin showed similar stoichiometries for the strong binding sites within the channels, but fewer weaker binding sites when compared to the H-chain protein. The kinetics and binding data indicate that the binding of Zn(II) and Tb(III) in the three-fold channels, which is the main pathway of iron(II) entry in ferritin, blocks the access of most of the iron to the ferroxidase sites on the interior of the protein, accounting for the strong inhibition by these metal ions of the oxidative deposition of iron in ferritin.     

Iron incorporation into apoferritin. The role of apoferritin as a ferroxidase

Apoferritin catalyzes the oxidation of Fe(II) to Fe(III). Ferroxidase activity is assayed and characterized by coupling the oxidation with the binding of Fe(III) to transferrin. The initial rate of Fe(II) oxidation is dependent on apoferritin and initial Fe(II) concentration but independent of transferrin concentration. The ferroxidase activity is inhibited by Zn(II). Ferritins with varying loads of iron have the same ferroxidase activity level. It is suggested that the described oxidation process represents the initial step of iron deposition in apoferritin. Since transferrin can intercept Fe(III) before it is deposited in apoferritin, active sites for Fe(II) oxidation must be on or near the surface of apoferritin. This finding is contrary to the current view of apoferritin-catalyzed oxidation of Fe(II) which places active sites in the channels to the core or inside the central core.     

Iron(III) clusters bound to horse spleen apoferritin: an X-ray absorption and M?ssbauer spectroscopy study that shows that iron nuclei can form on the protein

Ferritin is a complex of a hollow, spherical protein and a hydrous, ferric oxide core of less than or equal to 4500 iron atoms inside the apoprotein coat; the apoprotein has multiple (ca. 12) binding sites for monoatomic metal ions, e.g., Fe(II), V(IV), Tb(III), that may be important in the initiation of iron core formation. In an earlier study we observed that the oxidation of Fe(II) vacated some, but not all, of the metal-binding sites, suggesting migration of some Fe during oxidation, possibly to form nucleation clusters; some Fe(III) remained bound to the protein. Preliminary extended X-ray absorbance fine structure (EXAFS) analysis of the same Fe(III)-apoferritin complex showed an environment distinct from ferritin cores, but the data did not allow a test of the Fe cluster hypothesis. In this paper, with improved EXAFS data and with M?ssbauer data on the same complex formed with 57Fe, we clearly show that the Fe(III) in the distinctive environment is polynuclear (Fe atoms with Fe-Fe = 3.5 A and TB = 7 K). Moreover, the arrangement of atoms is such that Fe(III) atoms appear to have both carboxylate-like ligands, presumably from apoferritin, and oxo bridges to the other iron atoms. Thus the protein provides sites not only for initiation but also for nucleation of the iron core. Sites commodious enough and with sufficient conserved carboxylate ligands to accommodate such a nucleus occur inside the protein coat at the subunit dimer interfaces. Such Fe(III)-apoferritin nucleation complexes can be used to study the properties of the several members of the apoferritin family.     

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.     

Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin

Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV–vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein.     

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.     

Nonequivalence of the metal binding sites of conalbumin. Calorimetric and spectrophotometric studies of aluminum binding.

Differential scanning calorimetric experiments show that addition of Al(III) to conalbumin increases its denaturation temperature by 5 degrees, from 60 to 68 degrees. Only one Al(III) bound per conalbumin molecule produces this change in heat stability; additional bound Al(III) does not affect the heat stability. Since Al(III) displaces both Cu(II) bound at the metal binding sites of conalbumin, binding of aluminum takes place at the same metal binding sites. The binding constant for the second Al(III) is at least 100-fold less than that for the binding of the first Al(III), and both are displaced by added iron. The order of increasing heat stability of the metal ion complexes of conalbumin, Cu(II), Al(III), Fe(III), is the order of increasing binding constant for these metal ions.     

Application of Freundlich and Langmuir models to multistage purification process to remove heavy metal ions by using Schizomeris leibleinii

The adsorption of iron(III), lead(II) and cadmium(II) ions onto Schizomeris leibleinii, a green alga, was studied with respect to initial pH, temperature, initial metal ion and biomass concentration to determine the optimum adsorption conditions. Optimum initial pH for iron(III), lead(II) and cadmium(II) ions were 2.5, 4.5 and 5.0 at optimum temperature 30°C, respectively. The initial adsorption rates increased with increasing initial iron(III), lead(II) and cadmium(II) ion concentrations up to 100, 100 and 150 mg l⁻¹, respectively. The Freundlich and Langmuir adsorption isotherms were developed at various initial pH and temperature values. The adsorption of these metal ions to S. leibleinii was investigated in a two-stage mixed batch reactor. The residual metal ion concentrations (C_eq) at equilibrium at each stage for a given ‘quantity of dried algae (X₀)/volume of solution containing heavy metal ion (V₀)’ ratio were calculated using Freundlich and Langmuir isotherm constants. The experimental biosorption equilibrium data for iron(III), lead(II) and cadmium(II) ions were in good agreement with those calculated by both Freundlich and Langmuir models. The adsorbed iron(III), lead(II) and cadmium(II) ion concentrations increased with increasing X₀/V₀ ratios while the adsorbed metal quantities per unit mass of dried algae decreased.     

Redox reactions of apo mammalian ferritin.

Apo horse spleen ferritin undergoes a 6.3 +/- 0.5 electron redox reaction at -310 mV at pH 6.0-8.5 and 25 degrees C to form reduced apoferritin (apoMFred). Reconstituted ferritin containing up to 50 ferric ions undergoes reduction at the same potential, taking up one electron per ferric ion and six additional electrons by the protein. We propose that apo mammalian ferritin (apoMF) contains six redox centers that can be fully oxidized forming oxidized apoferritin (apoMFox) or fully reduced forming apoMFred. ApoMFred can be prepared conveniently by dithionite or methyl viologen reduction. ApoMFred is slowly oxidized by molecular oxygen but more rapidly by Fe(CN)6(3-) to apoMFox. Fe(III)-cytochrome c readily oxidizes apoMFred to apoMFox with a stoichiometry of 6 Fe(III)-cytochrome c per apoMFred, demonstrating a rapid interprotein electron-transfer reaction. Both redox states of apoMF react with added Fe3+ and Fe2+. Addition of eight Fe2+ to apoMFox under anaerobic conditions produced apoMFred and Fe3+, as evidenced by the presence of a strong g = 4.3 EPR signal. Subsequent addition of bipyridyl produced at least six Fe(bipyd)3(2+) per MF, establishing the reversibility of this internal electron-transfer process between the redox centers of apoMF and bound iron. Incubation of apoMFred with the Fe(3+)-ATP complex under anaerobic conditions resulted in the formation and binding of two Fe2+ and four Fe3+ by the protein. The various redox states formed by the binding of Fe2+ and Fe3+ to apoMFox and apoMFred are proposed and discussed. The yellow color of apoMF appears to be an integral characteristic of the apoMF and is possibly associated with its redox activity.     

The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins

Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.     

Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O(2). However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.     

Spectroscopic analysis of the unfolding of transition metal-ion complexes of human lactoferrin and transferrin.

1. Human lactoferrin and transferrin are capable of binding several transition metal ions [Fe(III), Cu(II), Mn(III), Co(III)] into specific binding sites in the presence of bicarbonate. 2. Increased conformational stability and increased resistance to protein unfolding is observed for these metal-ion complexes compared to the apoprotein form of these proteins. 3. Mn(III)-lactoferrin and transferrin complexes exhibit steeper denaturation transitions than the Co(III) complexes of these proteins suggesting greater cooperativity in the unfolding process. 4. The incorporation of Fe(III) into the specific metal binding sites offers the greatest resistance to thermal unfolding when compared to the other transition metal ions studied. 5. Non-coincidence of unfolding transitions is observed, with fluorescence transition midpoints being lower than those determined by absorbance measurements. 6. Fully denatured proteins in the presence of urea and alkyl ureas exhibit fluorescence wavelength maxima at 355-356 nm indicative of tryptophan exposure upon protein unfolding.     

Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase

Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Differential scanning calorimetry of aposuperoxide dismutase and metalated superoxide dismutase unfolding transitions has provided quantitative estimates of the metal binding affinities for manganese superoxide dismutase. The binding constant for Mn(II) (K(Mn(II)) = 3.2 x 10(8) m(-1)) is surprisingly low in light of the essentially irreversible metal binding characteristic of this family of proteins and indicates that metal binding and release processes are dominated by kinetic, rather than thermodynamic, constraints. The kinetic stability of the metalloprotein complex can be traced to stabilization by elements of the protein that are independent of the presence or absence of the metal ion reflected in the thermally triggered metalation characteristic of these proteins. Binding constants for Mn(III), Fe(II), and Fe(III) complexes were estimated using quasireversible values for the unfolding enthalpy and DeltaC(p) for apo-Mn superoxide dismutase and the observed T(m) values for unfolding the metalated species in the absence of denaturants. For manganese and iron complexes, an oxidation state-dependent binding affinity reflects the protein perturbation of the metal redox potential.     

Negative cooperativity of chicken ovotransferrin on Al(III)-binding

Chicken ovotransferrin, an iron binding protein, has two metal binding sites (amino (N) and carboxy (C) terminal sites). It binds Cu(II), Al(III), Co(II), and other metals, as well as Fe(III). In this study, the selectivity and cooperativity of the N and C sites on Al(III), Co(II), and Tb(III) binding were investigated. Metals were classified into two groups according to their site preference. Co(II) and Al(III) bound to the N site more preferably than to the C site, whereas Tb(III) bound to the C site more preferably. On Fe(III) binding, the binding constant of Fe(III) becomes larger when the other site is already occupied. Thus, positive cooperativity is seen. In the present study, the binding cooperativities of Co(II), Tb(III), and Al(III) as to the N and C sites were investigated. On Co(II) and Tb(III) binding, no cooperativity was observed, as in the case of Cu(II) [Yamamura, T. et al. (1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R.R., & Mazurier, J., eds.) pp. 53-56, Elsevier Science Publ. B.V., Amsterdam]. In contrast, negative cooperativity was observed on Al(III) binding. Based on a model proposed by Yamamura et al. [Yamamura, T. et al. (1985) ibid.], the ratio of the binding constants, KC/KN, and the stacking coefficient, Kst, were estimated. KC/KN is 2.2 +/- 0.4 for the Tb(III) ion, 0.5 +/- 0.1 for the Co(II) ion, and 0.12 +/- 0.02 for the Al(III) ion. Kst (= 1 in a non-cooperative case) is 0.98 +/- 0.02 for the Tb(III) ion, 1.03 +/- 0.02 for the Co(II) ion, and 0.55 +/- 0.22 for the Al(III) ion.     

Fe(III) and Cu(II) conalbumin visible circular dichroism spectra.

The single polypeptide chain of conalbumin strongly binds two Fe(III) or two Cu(II) ions to yield intense absorption in the visible region similar to that shown by the related protein transferrin. Comparison of the metal-ion-binding sites in the two proteins is made by exploiting the sensitivity to ligand geometry of circular dichroism (CD). For the Fe(III) proteins strong similarities of the CD spectra outweigh marginal differences. For Cu(II) conalbumin an additional negative extremum near 506 nm appears between two positive ones at 634 and 410 nm suggesting greater subtraction of oppositely signed CD components leading to lesser magnitudes for the two positive peaks than are found in Cu(II)-transferrin. The two Fe(III)-binding sites within conalbumin are compared by noting the strong similarities of the CD and MCD of proteins with Fe(III) in one site and Ga(III) in the other site, and vice versa, with the protein containing Fe(III) in both sites. Due to features of the amino acid sequences of the single protein chains, the four strong metal ion binding sites in conalbumin and transferrin cannot be identical in all particulars, yet CD spectra of their metal ion complexes are closely similar. From a study of model phenolate complexes and the wavelength maxima of visible absorption in the Fe(III), Cu(II), and Co(III) proteins near 465, 440, and 405 nm, respectively, these strong absorption bands are identified as ligand to metal ion electron-transfer transitions. It is suggested that tyrosyl residues are the donors in the electron transfer transitions and that they lock in the metal ions after being keyed into position by binding of bicarbonate or other anions.     

The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin

Despite previous detection of hydroxyl radical formation during iron deposition into ferritin, no reports exist in the literature concerning how it might affect ferritin function. In the present study, hydroxyl radical formation during Fe(II) oxidation by apoferritin was found to be contingent on the "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferritin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(II) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0, was approximately 3.1 Fe(II)/O(2) at all iron-to-protein ratios tested. The addition of HEPES as an alternate reactant for the hydroxyl radical resulted in a stoichiometry of about 2 Fe(II)/O(2) at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for its omission from reaction mixtures containing Fe(II) and apoferritin resulted in alterations to the ferritin consistent with oxidative damage. The kinetic parameters for the reaction of recombinant human H apoferritin with Fe(II) in HEPES buffer (100 mM) were: K(m) = 60 microM, k(cat) = 10 s(-1), and k(cat)/K(m) = 1.7 x 10(5) M(-1) x (-1). Collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adequate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro using nonphysiologic conditions. The possibility that these findings may have physiological significance is discussed.