Site-directed mutagenesis and biochemical analysis of the endogenous ligands in the ferrous active site of clavaminate synthase. The His-3 variant of the 2-His-1-carboxylate model

The facial 2-His-1-carboxylate (Asp/Glu) motif has emerged as the structural paradigm for metal binding in the alpha-ketoglutarate (alpha-KG)-dependent nonheme iron oxygenases. Clavaminate synthase (CS2) is an unusual member of this enzyme family that mediates three different, nonsequential reactions during the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In this study, covalent modification of CS2 by the affinity label N-bromoacetyl-L-arginine near His297, which is within the HRV signature of a His-2 motif, suggested this histidine could play a role in metal coordination. However, site-specific mutagenesis of eight His residues to Gln identified His145 and His280, but not His297, as involved in iron binding. Weak homology of His145 and its flanking sequence and the presence of Glu147 fitting the canonical acidic residue of the His-Xaa-Asp/Glu signature are consistent with His145 being a coordinating ligand (His-1). His280 and its flanking sequence, which give poor alignments to most other members of this enzyme family, are similar among a subset of these enzymes and notably to CarC, an apparent oxygenase involved in carbapenem biosynthesis. The separation of His145 and His280 is more than twice that seen in the current 2-His-1-carboxylate model and may define an alternative iron binding motif, which we propose as His-3. These ligand assignments, based on kinetic measurements of both oxidative cyclization/desaturation and hydroxylation assays, establish that no histidine ligand switching occurs during the catalytic cycle. These results are confirmed in a recent X-ray crystal structure of CS1, a highly similar isozyme of CS2 (81% identical). Tyr299, Tyr300 in CS2 modified by N-bromoacetyl-L-arginine, is hydrogen bonded to Glu146 (Glu147 in CS2) in this structure and well-positioned for reaction with the affinity label.     

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.     

Metal-catalyzed oxidation reactions and mass spectrometry: the roles of ascorbate and different oxidizing agents in determining Cu-protein-binding sites

Further study has been made of metal-catalyzed oxidation (MCO) reactions and mass spectrometry as a method to determine the binding site of copper in metalloproteins. The role of ascorbate and a variety of oxidizing agents, including O2, H2O2, and S2O8(2-), have been investigated using Cu/Zn superoxide dismutase (SOD) as a model system. Ascorbate is found to play two competing roles in the MCO reactions. It reduces Cu(II), which initiates and maintains the generation of reactive oxygen species, and it scavenges radicals, which helps to localize oxidation products to amino acids near the metal center. An ascorbate concentration of 100 mM is found to be optimal with regard to localizing oxidation products to only the Cu-binding residues (His44, His46, His61, and His118) of Cu/Zn SOD. This concentration of ascorbate is very similar to the optimum concentration found in our previous studies of different Cu-binding proteins. Another notable result from this study is the observation that S2O8(2-) is more effective as an oxidant than O2 or H2O2 in the MCO reactions. Because S2O8(2-) is more stable in solution than H2O2, using it as an oxidizing agent results in much less nonspecific oxidation to the protein. The overall results of this study suggest that general MCO reaction conditions may exist for determining the metal-binding site of a wide range of Cu-binding proteins.     

Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits

Amyloid beta peptide (Abeta) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimer's disease. Cu(2+) and Zn(2+) bind Abeta, inducing aggregation and giving rise to reactive oxygen species. These reactions may play a deleterious role in the disease state, because high concentrations of iron, copper, and zinc have been located in amyloid in diseased brains. Here we show that coordination of metal ions to Abeta is the same in both aqueous solution and lipid environments, with His(6), His(13), and His(14) all involved. At Cu(2+)/peptide molar ratios >0.3, Abeta coordinated a second Cu(2+) atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at N(epsilon)2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu(2+) or Zn(2+) to Abeta in a negatively charged lipid environment caused a conformational change from beta-sheet to alpha-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Abeta generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.     

Kinetic analysis of the role of histidine chloramines in hypochlorous acid mediated protein oxidation

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and chloride ions by the heme enzyme myeloperoxidase (MPO) released from activated leukocytes. In addition to its potent antibacterial effects, excessive HOCl production can lead to host tissue damage, with this implicated in human diseases such as atherosclerosis, cystic fibrosis, and arthritis. HOCl reacts rapidly with biological materials, with proteins being major targets. Chlorinated amines (chloramines) formed from Lys and His side chains and alpha-amino groups on proteins are major products of these reactions; these materials are however also oxidants and can undergo further reactions. In this study, the kinetics of reaction of His side-chain chloramines with other protein components have been investigated by UV/visible spectroscopy and stopped flow methods at pH 7.4 and 22 degrees C, using the chloramines of the model compound 4-imidazoleacetic acid and N-alpha-acetyl-histidine. The second-order rate constants decrease in a similar order (Cys > Met > disulfide bonds > Trp approximately alpha-amino > Lys > Tyr > backbone amides > Arg) to the corresponding reactions of HOCl, but are typically 5-25 times slower. These rate constants are consistent with His side-chain chloramines being important secondary oxidants in HOCl-mediated damage. These studies suggest that formation and subsequent reactions of His side-chain chloramines may be responsible for the targeted secondary modification of selected protein residues by HOCl that has previously been observed experimentally and highlight the importance of chloramine structure on their subsequent reactivity.     

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.     

On the role of the axial ligand in heme proteins: a theoretical study

We present a systematic investigation of how the axial ligand in heme proteins influences the geometry, electronic structure, and spin states of the active site, and the energies of the reaction cycles. Using the density functional B3LYP method and medium-sized basis sets, we have compared models with His, His+Asp, Cys, Tyr, and Tyr+Arg as found in myoglobin and hemoglobin, peroxidases, cytochrome P450, and heme catalases, respectively. We have studied 12 reactants and intermediates of the reaction cycles of these enzymes, including complexes with H(2)O, OH(-), O(2-), CH(3)OH, O(2), H(2)O(2), and HO(2)(-) in various formal oxidation states of the iron ion (II to V). The results show that His gives ~0.6 V higher reduction potentials than the other ligands. In particular, it is harder to reduce and protonate the O(2) complex with His than with the other ligands, in accordance with the O(2) carrier function of globins and the oxidative chemistry of the other proteins. For most properties, the trend Cys相似文献   

Structure and spatial conformation of the iron-binding sites of transferrins

Transferrins are iron-binding glycoproteins involved in iron metabolism and antibacterial defense mechanisms. Since the discovery of transferrins, many studies have attempted to characterize the iron ligands and to establish the conformation of the iron-binding sites. From chemical and spectroscopic studies, it was generally accepted that iron was hexacoordinated to Tyr and His residues, to a water molecule and to a (bi)carbonate ion, electrostatically linked to an Arg residue. On the basis of these studies, on the one hand, and on the basis of the homologies between the amino acid sequences of transferrins, on the other hand, predicted data have been provided about the number and location of the iron ligands. Recent X-ray crystallography studies of human lactotransferrin have partially confirmed the above-mentioned predicted data and have brought invaluable information about the nature of the ligands and the conformation of the iron-binding site. On the basis of the obtained results, a scheme has been proposed in which the iron is coordinated to 2 Tyr, 1 His and 1 Asp residues, to a (bi)carbonate linked to an Arg residue and probably to a water molecule. The iron-binding site is located at the interface between the two domains which constitute each lobe of the transferrins.     

Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

The iron atom in the nonheme iron monooxygenase phenylalanine hydroxylase is bound on one face by His285, His290, and Glu330. This arrangement of metal ligands is conserved in the other aromatic amino acid hydroxylases, tyrosine hydroxylase and tryptophan hydroxylase. A similar 2-His-1-carboxylate facial triad of two histidines and an acidic residue are the ligands to the iron in other nonheme iron enzymes, including the α-ketoglutarate-dependent hydroxylases and the extradiol dioxygenases. Previous studies of the effects of conservative mutations of the iron ligands in tyrosine hydroxylase established that there is some plasticity in the nature of the ligands and that the three ligands differ in their sensitivity to mutagenesis. To determine the generality of this finding for enzymes containing a 2-His-1-carboxylate facial triad, the His285, His290, and Glu330 in rat phenylalanine hydroxylase were mutated to glutamine, glutamate, and histidine. All of the mutant proteins had low but measurable activities for tyrosine formation. In general, mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All of the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. The H285Q enzyme had the highest activity as tetrahydropterin oxidase at 20% the wild-type value. All of the mutations greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.     

The kinetics and mechanisms of the complex formation and antioxidant behaviour of the polyphenols EGCg and ECG with iron(III)

(-)-Epigallocatechin-gallate ((-)-EGCg) and (-)-epicatechin-gallate ((-)-ECG) are important antioxidants which are found in green tea. The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with EGCg and ECG have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are consistent with a mechanism in which the 2:1 metal:ligand complex initially formed on reaction of iron(III) with the ligand subsequently decomposes in an electron transfer step. Complex formation takes place at two separate binding sites via coupled reactions. Rate constants of 4.28(+/-0.06) x 10(6) M(-2) s(-1) and 2.83(+/-0.04) x 10(6) M(-2) s(-1) have been evaluated for the reaction of monohydroxy Fe(OH)2+ species with EGCg and ECG, respectively while rate constants for of 2.94(+/-0.4) x 10(4) M(-2) s(-1) and 2.41(+/-0.25) x 10(4) M(-2) s(-1) have been evaluated for the reaction of Fe3+ species with EGCg and ECG, respectively. The iron(III) assisted decomposition of the initial iron(III) complex formed was also investigated and the rate constants evaluated. Both the complex formation and subsequent electron transfer reactions of iron(III) with EGCg and ECG were monitored using UV-visible spectrophotometry. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time dependant spectra. The results obtained show that one molecule of either EGCg or EGC is capable of reducing up to four iron(III) species, a fact which is consistent with the powerful antioxidant properties of the ligands.     

Site-directed mutants of human myeloperoxidase. A topological approach to the heme-binding site.

Two site-directed mutants of human promyeloperoxidase, MPO(His416----Ala) and MPO(His502----Ala), have been expressed in Chinese hamster ovary cells and purified. Overall purification yields and apparent molecular masses of the mutant proteins were similar to those of the wild-type enzyme. Both mutant species were analyzed spectroscopically to check the presence of the hemic iron in the proteins and were assayed for peroxidase activity. The data show that substitution of His502 leads to the loss, or to an inappropriate configuration, of the heme together with the loss of activity, suggesting that this residue could be the proximal His involved in the binding to the iron centers. On the other hand, substitution of His416 by alanine had no effect on either of the studied parameters.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.     

The kinetics and mechanisms of the reactions of iron(III) with quercetin and morin

The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with the flavonoids quercetin and morin have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M. Mechanisms have been proposed which account satisfactorily for the kinetic data. The data are consistent with a mechanism in which the metal:ligand complex formed initially on reaction of iron(III) with the ligand subsequently decomposes through an electron transfer step. Morin forms a 1:1 metal:ligand complex while quercetin forms a 2:1 metal:ligand complex. Both ligands showed evidence for the involvement of the iron hydroxo dimer Fe2(OH)2(4+) in the complex formation reaction at the hydroxy-carbonyl moiety. The iron(III) assisted decomposition of the initial iron(III) complex formed was also investigated and the rate constants evaluated. Both the complex formation and subsequent electron transfer reactions of iron(III) with these ligands were monitored using UV-visible spectrophotometry. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time dependant spectra.     

Structure-function studies of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron-dependent oxygenase that catalyzes the ring closure of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies and the crystal structure of IPNS show that the iron atom in the active species is coordinated to two histidine and one aspartic acid residues, and to ACV, dioxygen and H2O. We previously showed by site-directed mutagenesis that residues His212, Asp214 and His268 in the IPNS of Streptomyces jumonjinensis are essential for activity and correspond to the iron ligands identified by crystallography. To evaluate the importance of the nature of the protein ligands for activity, His214 and His268 were exchanged with asparagine, aspartic acid and glutamine, and Asp214 replaced with glutamic acid, histidine and cysteine, each of which has the potential to bind iron. Only the Asp214Glu mutant retained activity, approximately 1% that of the wild type. To determine the importance of the spatial arrangement of the protein ligands for activity, His212 and His268 were separately exchanged with Asp214; both mutant enzymes were completely defective. These findings establish that IPNS activity depends critically on the presence of two histidine and one carboxylate ligands in a unique spatial arrangement within the active site. Molecular modeling studies of the active site employing the S. jumonjinensis IPNS crystal structure support this view. Measurements of iron binding by the wild type and the Asp214Glu, Asp214His and Asp214Cys-modified proteins suggest that Asp214 may have a role in catalysis as well as in iron coordination.     

Steady magnetic fields effect on lipid peroxidation kinetics

The effect of steady magnetic fields (ranging from 0 to 280 mT) has been investigated on the kinetics of non-enzymatic lipid peroxidation occurring in a model system consisting of liposomes obtained from 1, 2-dioleoylphosphatidylcholine by oxygen consumption. The process was found to be accelerated by weak steady magnetic fields. A computer simulation method was employed to detect the reactions that govern the process kinetics, to elucidate magneto-sensitive stages (initiation and reduction of iron(III), as well as lipid peroxide radical recombination) and to determine their rate constants at various external magnetic fields. The kinetics of peroxidation of lipid cell membranes have been modeled mathematically at oxygen and ‘free’ iron concentrations close to those in the cells and also at increased free iron concentrations at different external magnetic field values.     

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) > His39Cys > His39Ser.     

Hydrogen-deuterium exchange in the histidine residues of bovine alpha-lactalbumin.

The proton magnetic resonance spectrum of bovine alpha-lactalbumin has been observed, and three peaks assignable to the position-2 CH protons of the three histidine rpsidues (His 32, 68, and 107) of this protein have been subjected to detailed examination. The assignments of these peaks to His 32, 68, and 107 were made on the basis of the difference in their reactivities with iodoacetic acid. The rate constants of the hydrogen-deuterium exchange reactions were found to be 8.0 X 10(-5), 2.6 X 10(-4), and 8.0 X 10(-5) min-1, respectively, at pH 8.5 and at 35 degrees, while at 62 degrees all three were found to be 0.84 approximately 1.1 X 10(-2) min-1. On the basis of these data, it has been shown that, in the native form of this protein, His 68 is the most exposed to the solvent while His 32 and His 107 are buried slightly deeper in the surface of the molecule. The fluctuation amplitudes gamma, or the effective chances of His 32, 68, and 107 to be fully exposed to the solvent, were found to be 0.4, 1.3, and 0.4, respectively.     

Histidine ligand variants of a flavo-diiron protein: effects on structure and activities

Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O₂R). While most FDPs show similar structural, spectroscopic, and redox properties, O₂R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O₂R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and ?A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O₂R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O₂ were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O₂R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O₂R and NOR mechanisms of FDPs.     

2-Hydroxypyridine-N-oxides: effective new chelators in iron mobilisation

The 2-hydroxypyridine-N-oxide derivatives, 2-hydroxypyridine-N-oxide, 2,4-dihydroxypyridine-N-oxide, 2-hydroxy-4-methoxypyridine-N-oxide and 2-hydroxy-4-(2'-methoxyethoxy)pyridine-N-oxide have been shown to remove iron from human transferrin and horse spleen ferritin at pH 7.4 at levels higher than those caused by desferrioxamine. Their reactions with transferrin were mainly biphasic and took 2-5 h to reach completion but iron mobilisation from ferritin was slower and their reactions continued after 40 h of incubation. The intraperitoneal and intragastric administration of 2,4-dihydroxypyridine-N-oxide to two iron-loaded 59Fe-labelled mice caused an increase in 59Fe excretion which is comparable to that caused by desferrioxamine intraperitoneally. These results increase the prospects for the use of these chelators as probes for studying iron metabolism and in the treatment of iron overload and other diseases of iron imbalance.