Reaction of oxygen with 6-hydroxydopamine catalyzed by Cu, Fe, Mn, and V complexes: identification of a thermodynamic window for effective metal catalysis

In the autoxidation of 6-hydroxydopamine, we investigated the reactivity of metals and metal complexes with a range of abilities to catalyse the reaction with oxygen. Comparing the catalytic effectiveness of aquo metals at pH 7.4, copper accelerated autoxidation 61-fold, iron 24-fold, manganese 7.3-fold, and vanadium 5.7-fold. Copper was thus the most effective catalyst despite being the weakest oxidant, indicating reduction of oxygen as rate limiting. EDTA, which decreases the reduction potential of Fe(III)/Fe(II), increased catalysis by iron 74% to almost that of aquo copper. Conversely, EDTA inhibited catalysis by copper, manganese, and vanadium. Desferrioxamine strongly inhibited catalysis by all of the metals. Histidine prevented catalysis by copper, accelerated catalysis by iron (43%), and had little effect on catalysis by manganese or vanadium. ADP and phytate inhibited catalysis by iron and manganese (50% or more), accelerated catalysis by vanadium (10-27%), and had no effect on catalysis by copper. The effects of the ligands largely reflected their influence on the reduction potential of the metal. Accordingly, addition of NaBr, which increases the reduction potential of Cu(II)/Cu(I), inhibited by 50%. In contrast, Na2SO4 augmented catalysis by copper 3-fold. Consistent with effects of OH- on reduction potentials and on metal coordination to 6-hydroxydopamine, an increase in pH to 8.0 decreased catalysis by copper and iron, but increased that of manganese 10-fold. In conclusion, the catalytic effectiveness of the metal-ligand complexes are largely attributable to their reduction potential, with steric accessibility playing secondary roles. The results delineate a window of catalytically effective potentials suitable for facile reduction and reoxidation by oxygen. By extension the results identify factors determining the pro- and antioxidant roles of ligands in metal mediated reduction of oxygen.     

Di- and trinuclear arrangements of zinc(II)-1,5,9-triazacyclododecane units on the calix[4]arene scaffold: Efficiency and substrate selectivity in the catalysis of ester cleavage

The catalytic activity of the zinc(II) complexes of calix[4]arenes decorated with 1,5,9-triazacyclododecane ligands at the 1,2-, 1,3-, and 1,2,3-positions of the upper rim was investigated in the basic methanolysis (pH 10.4) of aryl acetates functionalised at the meta- and para-positions with a carboxylate anchoring group. Michaelis-Menten kinetics and turnover catalysis were observed. High rate accelerations, up to more than 10⁴-fold at 0.2 mM catalyst, were recorded in the most favourable catalyst-substrate combinations. The order of catalytic efficiency of regioisomeric bimetallic complexes is 1,2-vicinal ? 1,3-distal, resulting from a significant degree of synergism between metal ions in the former, and a complete lack in the latter. The moderately higher efficiency of the trimetallic compared with the 1,2-vicinal bimetallic catalyst provides an indication of a possible cooperation of three zinc(II) ions in the catalysis.     

Dopamine complexes of iron in the etiology and pathogenesis of Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimers. The main pathological hallmark of Parkinson’s is the deterioration and death of neurons that produce the neurotransmitter dopamine. Much of the neuronal damage takes place in the substantia nigra, a small region of the midbrain that contains the cell bodies of neurons that produce dopamine. The deterioration and death of dopaminergic neurons are directly associated with misfolding and aggregation of proteins, principally α-synuclein, that are natively unfolded. Present also in the substantia nigra is an unusually high concentration of vestigial iron. Protein misfolding in non-genetic (sporadic) cases of PD has been associated with reactive oxygen species formed as products of O₂ reduction by the combination of dopamine and iron. Combinations of Fe³⁺, dopamine hydrochloride (DA^H+Cl), and various ancillary ligands have been studied as a function of pH in aqueous solution to determine the optimum pH for complex formation. With ancillary ligands (L₄) derived from nitrilotriacetic acid and ethylenediamine diacetic acid spectral changes are consistent with the formation of L₄Fe(DA^H+) species that reach a maximum concentration at pH 7.2. With edta as the ancillary ligand, spectral features at pH 7 resemble those of Fe³⁺-catecholate complexes that contain catecholate ligands bonded through a single oxygen. This demonstrates the ability of the dopamine catechol functionality to penetrate the coordination sphere of even exceptionally stable iron chelates.     

Exchange of iron by gallium in siderophores

Siderophores are iron transport compounds produced by numerous microorganisms and which strongly chelate Fe(III), but not Fe(II). Other trivalent metals, such as Al(III), Cr(III), or Ga(III), are not capable of significantly displacing iron from siderophores. However, I demonstrate here that Ga(III) can effectively displace iron under reducing conditions. With ascorbate as reductant and ferrozine as Fe(II) trapping agent, the kinetics of reductive displacement of iron by Ga(III) were followed spectroscopically by the increase of absorbance at 562 nm due to formation of the Fe(II)-ferrozine complex. No significant reduction of siderophore occurred in the absence of Ga(III). With excess Ga(III), the displacement was quantitative and very rapid. The rate of metal exchange was pseudo first order with respect to Ga(III) concentration and highly pH dependent, suggesting that siderophore ligands are displaced from the iron in a concerted mechanism by Ga(III) and protonation to expose the Fe(III) to reduction by ascorbate. Reaction rates were dependent upon the structure of the siderophore, being greatest for ferric rhodotorulic acid and slowest for ferrichrome A at pH 5.4. The pH profile for ferric rhodotorulic acid was unusual in that it showed a maximum at pH 6.5, while all other siderophores examined showed an increase in rate as pH was lowered from 7.0. The physiological significance of this reaction to the clinical use of gallium is discussed.     

Activity of maize leaf phosphoenolpyruvate carboxylase in relation to tautomerization and nonenzymatic decarboxylation of oxaloacetate

The keto form of oxaloacetate (OAA), a product of phosphoenolpyruvate carboxylase (PEPC) activity, can undergo various nonenzymatic conversions which make conventional methods of assaying the enzyme difficult, because the products may either act as inhibitors or go undetected. In studies with PEPC isolated from leaves of maize, an assay coupled with reduction of OAA to malate was compared with product analysis using high-performance liquid chromatography and an assay based on Pi release. The results show that activity of the enzyme in the assay coupled to malate dehydrogenase is underestimated, to varying extents, depending on magnesium concentration, buffer, and pH. In the assay coupled to malate dehydrogenase, inaccuracies occur due to conversion of the keto form of OAA to the enol form, which is not utilized as a substrate, and due to loss of OAA by decarboxylation to pyruvate. The assay based on Pi formation is considered to give the true rate of catalysis. With this assay the pH optimum is 7.8, compared to 8.3-8.5 for the assay coupled to malate dehydrogenase. The metal enol complex of oxaloacetate (M-OAAenol) is an inhibitor of PEPC and conditions which are favorable for forming this tautomer, high pH with divalent metal ions or high concentrations of Tris buffer at a pH below its pKa value, limit catalysis. Glycine stimulates enzyme activity, and it may have its effect by preventing the formation of the hydrated M-OAAenol complex and maintaining more of the OAA in the keto form. This interpretation is consistent with glycine stimulation of malate synthesis in the assay of PEPC coupled to malate dehydrogenase, with glycine stimulation of the decarboxylation of OAA, and with a reduction in the level of the M-OAAenol complex in the presence of glycine.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.     

Catalysis of superoxide dismutation by manganese aminopolycarboxylate complexes

Complexes of manganese, copper, cobalt, and iron with a variety of aminopolycarboxylates at concentrations from 2 X 10(-7) to 3 X 10(-6) M were tested for superoxide dismutase activity with horse ferricytochrome c as the competing reagent for superoxide. In the presence of excess ligand only manganous nitrilotriacetate and manganous ethylenediaminediacetate showed activity with catalytic rate constants of 2.2 X 10(7) and 1.8 X 10(7) M-1 S-1, respectively, at pH 6, 22 +/- 1 degree C, and 10 mM ionic strength. These rate constants decrease considerably at higher pH. Manganous N-hydroxyethylethylenediaminetriacetate is oxidized by superoxide, but does not appear to have catalytic activity. From the experimental conditions under which the two complexes mentioned above exhibit catalysis, and the inactivity of other metal chelates, it is concluded that an open coordination site is essential but not sufficient to catalyze the dismutation reaction.     

A new thioether-ligated iron porphyrin as a model of a protonated form of P450 active site

Thioether-ligated iron porphyrin (complex 1) was synthesized as a model of the protonated form of P450 to explore the possible involvement of the protonated form in the catalytic cycle, and ether-ligated iron porphyrin (complex 2) was also synthesized for comparison. The thioether and ether ligands enhanced heterolytic O-O bond cleavage of peroxy acid-iron porphyrin complex even in highly hydrophobic media without the assistance of acid or base, using mCPPAA as an oxidant. Competitive oxidation of cyclooctane/cyclooctene catalyzed by iron porphyrins showed that complexes 1 and 2 are less effective than heme thiolate (P450 and a synthetic heme thiolate (SR complex)) in oxidizing alkane. The possibility that thiol-ligated heme, which is a protonated form of heme thiolate, is not involved in the active intermediate structure of P450 is indicated by this result. This is the first report concerning the oxidizing ability of a thioether-ligated iron porphyrin.     

The mechanisms of S-nitrosothiol decomposition catalyzed by iron.

The mechanisms of S-nitrosothiol transformation into paramagnetic dinitrosyl iron complexes (DNICs) with thiol- or non-thiol ligands or mononitrosyl iron complex (MNICs) with N-methyl-D-glucamine dithiocarbamate catalyzed by iron(II) ions under anaerobic conditions were studied by monitoring EPR or optical features of the complexes and S-nitrosothiols. The kinetic investigations demonstrated the appearance of short-living paramagnetic mononitrosyl-iron complex with L-cysteine prior to the formation of stable dinitrosyl-iron complex with cysteine in the solution of iron(II)-citrate complex (50-100 microM), S-nitrosocysteine (400 microM), and L-cysteine (20 mM) in 100 mM Hepes buffer (pH 7.4). The addition of deoxyhemoglobin (100 microM) did not influence the process, which points to a direct interaction between S-nitrosocysteine and iron(II) ions to yield DNIC. The reaction of DNIC-cysteine formation is first- and second-order in iron and S-nitrosocysteine, respectively. The third-order rate constant is (1.0 +/- 0.2) x 10(5) M(-2) s(-1) (estimated from EPR results) or (2.0 +/- 0.1) x 10(4) M(-2) s(-1) (estimated by optical method). A similar process of DNIC-cysteine formation was observed in a solution of iron(II)-citrate complex, L-cysteine, and NO-proline (200 microM) as a NO* donor. The appearance of a less stable dinitrosyl-iron complex with phosphate was detected when solutions of iron(II)-citrate containing 100 mM phosphate buffer (pH 7.4) were mixed with S-nitrosocysteine or NO-proline. The rapid formation of DNIC with phosphate was followed by its decay. When the concentration of L-cysteine in solutions was reduced from 20 to 1 mM, the life-time of the DNIC-cysteine diminished notably; this was caused by consumption of L-cysteine in the process of DNIC-cysteine formation from S-nitrosocysteine and iron. Thus, L-cysteine is consumed. Formation of DNIC with glutathione was also observed in a solution of glutathione (20 mM), S-nitrosoglutathione (400 microM), and iron(II) complex (800 microM) in 100 mM Hepes buffer (pH 7.4), but the rate of formation was about 10 times slower than the formation of the DNIC-cysteine. The rate of MNIC-MGD formation from iron(II)-MGD complexes and S-nitrosocysteine was first-order in both reactants. The second-order rate constant for this reaction, estimated from EPR measurements, was 30 +/- 5 M(-1) s(-1). Rate constants of MNIC-MGD formation from iron(II)-MGD and the more stable S-nitrosoglutathione and S-nitroso-D,L-penicillamine were equal to 3.0 +/- 0.3 and 0.3 +/- 0.05 M(-1) s(-1), respectively. Thus, the concerted mechanism of DNIC and MNIC formation from S-nitrosothiols and iron(II) ions can be suggested to be predominant.     

Inhibition of the purified 20S proteasome by non-heme iron complexes

Polypyridyl pentadentate ligands N4Py (1) and Bn-TPEN (2), along with their respective iron complexes, have been investigated for their ability to inhibit the purified 20S proteasome. Results demonstrated that the iron complexes of both ligands are potent inhibitors of the 20S proteasome (IC(50) = 9.2 μM for [Fe(II)(OH(2))(N4Py)](2+) (3) and 4.0 μM for [Fe(II)(OH(2))(Bn-TPEN)](2+) (4)). Control experiments showed that ligand 1 or Fe(II) alone showed no inhibition, whereas 2 was moderately active (IC(50) = 96 μM), suggesting that iron, when bound to these ligands, plays a key role in proteasome inhibition. Results from time-dependent inactivation studies suggest different modes of action for the iron complexes. Time-dependent decay of proteasome activity was observed upon incubation in the presence of 4, which accelerated in the presence of DTT, suggesting reductive activation of O(2) and oxidation of the 20S proteasome as a mode of action. In contrast, loss of 20S proteasome activity was not observed with 3 over time, suggesting inhibition through direct binding of the iron complex to the enzyme. Inhibition of the 20S proteasome by 4 was not blocked by reactive oxygen species scavengers, consistent with a unique oxidant being responsible for the time-dependent inhibition observed.     

Ferric iron complexes of dopamine and 5,6-dihydroxyindole with nta, edda, and edta as ancillary ligands

Coordination of the neurotransmitter dopamine (DA) and the metal-binding component of neuromelanin 5,6-dihydroxyindole (DHI) with ferric iron has been studied in aqueous solution in the presence of ancillary ligands containing amine nitrogen and carboxylate oxygen donor sites. With nitrilotriacetic acid (nta) and ethylenediamine diacetic acid (edda) coligands, coordination of the catecholate ligands DA and DHI is observed to be complete at physiological pH. The resulting complexes of DA have the characteristic two-component electronic spectrum observed characteristically for L₄Fe(Cat) complexes. The spectrum obtained with DHI consists of a single broad absorption in the visible region. Both DA and DHI are able to coordinate with Fe³⁺ in the presence of edta, displacing carboxylate oxygen donors at pH values just above physiological pH. These results demonstrate the strong affinity of DA and DHI for Fe³⁺, pointing to in vivo complex formation in neuronal mixtures at physiological pH.     

Ferric iron reduction by sulfur- and iron-oxidizing bacteria.

Acidophilic bacteria of the genera Thiobacillus and Sulfolobus are able to reduce ferric iron when growing on elemental sulfur as an energy source. It has been previously thought that ferric iron serves as a nonbiological oxidant in the formation of acid mine drainage and in the leaching of ores, but these results suggest that bacterial catalysis may play a significant role in the reactivity of ferric iron.     

Calcium supports loop closure but not catalysis in Rubisco

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses CO(2) assimilation in biology. A prerequisite for catalysis is an activation process, whereby an active site lysine is selectively carbamylated. The carbamyl group is then stablised by a metal ion, which in vivo is Mg(2+). Other divalent metal ions can replace Mg(2+) as activators in vitro, but the nature of the metal ion strongly influences the catalytic activity of the enzyme and has a differential effect on the ratio of the carboxylation reaction and the competing oxygenation reaction. Biochemical studies show that calcium promotes carbamylation but not catalysis. To investigate the role of the metal in catalysis, we have determined two structures of the enzyme complexed with Ca(2+) and the transition state analogue 2-carboxy-D-arbinitol-1,5-bisphosphate (2CABP). One of the complexes was prepared by soaking 2CABP into crystals of the enzyme-Ca(2+)-product complex, while the other was obtained by cocrystallising the enzyme with calcium and 2CABP under activating conditions. The two crystals belong to different space groups, and one was merohedrally twinned. Both complexes show very similar three-dimensional features. The enzyme is carbamylated at Lys201, and requisite loops close over the bound ligands in the active site, shielding them from the solvent in a manner similar to the corresponding complex with Mg(2+). However, there are subtle differences that could explain the particular role of Ca(2+) in these processes. The larger radius of the calcium ion and its reduced Lewis-acid character causes a significant increase in the required proton hop distance between the C3 proton and the carbamate on Lys201 in the calcium complex. This alone could explain the inability of calcium to sustain catalysis in Rubisco. Similar effects are also expected on subsequent proton transfer steps in the catalytic cycle. Here we also discuss the effect of metal substitution on the dynamics of the ligands around the metal ion.     

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.     

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.     

N-hydroxyguanidines as new heme ligands: UV-visible, EPR, and resonance Raman studies of the interaction of various compounds bearing a C=NOH function with microperoxidase-8

Interaction between microperoxidase-8 (MP8), a water-soluble hemeprotein model, and a wide range of N-aryl and N-alkyl N'-hydroxyguanidines and related compounds has been investigated using UV-visible, EPR, and resonance Raman spectroscopies. All the N-hydroxyguanidines studied bind to the ferric form of MP8 with formation of stable low-spin iron(III) complexes characterized by absorption maxima at 405, 535, and 560 nm. The complex obtained with N-(4-methoxyphenyl) N'-hydroxyguanidine exhibits EPR g-values at 2.55, 2.26, and 1.86. The resonance Raman (RR) spectrum of this complex is also in agreement with an hexacoordinated low-spin iron(III) structure. The dissociation constants (K(s)) of the MP8 complexes with mono- and disubstituted N-hydroxyguanidines vary between 15 and 160 microM at pH 7.4. Amidoximes also form low-spin iron(III) complexes of MP8, although with much larger dissociation constants. Under the same conditions, ketoximes, aldoximes, methoxyguanidines, and guanidines completely fail to form such complexes with MP8. The K(s) values of the MP8-N-hydroxyguanidine complexes decrease as the pH of the solution is increased, and the affinity of the N-hydroxyguanidines toward MP8 increases with the pK(a) of these ligands. Altogether these results show that compounds involving a -C(NHR)=NOH moiety act as good ligands of MP8-Fe(III) with an affinity that depends on the electron-richness of this moiety. The analysis of the EPR spectrum of the MP8-N-hydroxyguanidine complexes according to Taylor's equations shows a strong axial distortion of the iron, typical of those observed for hexacoordinated heme-Fe(III) complexes with at least one pi donor axial ligand (HO(-), RO(-), or RS(-)). These data strongly suggest that N-hydroxyguanidines bind to MP8 iron via their oxygen atom after deprotonation or weakening of their O-H bond. It thus seems that N-hydroxyguanidines could constitute a new class of strong ligands for hemeproteins and iron(III)-porphyrins.     

Effect of pH on the liver alcohol dehydrogenase reaction.

New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.     

Fluorescence quenching and bonding properties of some hydroxamic acid derivatives by iron(III) and manganese(II)

Spectrophotometric investigations of highly fluorescent metal chelating molecules are of relevance due to their potential application in novel, selective fluorescence‐based sensors. Benzene and naphthalene chromophores are highly fluorescent while hydroxamic acids are widely used as ligands for complexation of transition metals. In order to develop fluorescence probes, several phenyl derivatives of N‐phenylbenzohydroxamic acid and an aminodihydroxamic acid linked with a naphthalene chromophore were synthesized and their selective ionophoric properties towards iron(III) and manganese(II) ions were investigated using fluorescence and absorption spectroscopy. Both methods confirm the formation of 1:1 and 1:2 complexes for iron(III) and a 1:1 complex for manganese(II). The complex that is formed depends on the concentration of the ligand and pH of the medium. The amino dihydroxamic acid exhibits a prominent selectivity towards iron(III) with a two‐step 1:1 and 1:2 quenching mechanism at pH 3 and towards manganese(II) with a 1:1 quenching mechanism at a probe concentration of 1 × 10^?5 mol dm^?3 at pH 9.5 The logarithm of overall formation constants of 1:1 and 1:2 complexes of iron(III) were estimated as 3.30 and 9.05, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Ferrous ion autoxidation and its chelation in iron-loaded human liver HepG2 cells.

Ferrous ion (Fe(2+)) is long thought to be the most likely active species, producing oxidants through interaction of Fe(2+) with oxygen (O(2)). Because current iron overload therapy uses only Fe(3+) chelators, such as desferrioxamine (DFO), we have tested a hypothesis that addition of a Fe(2+) chelator, 2,2'-dipyridyl (DP), may be more efficient and effective in preventing iron-induced oxidative damage in human liver HepG2 cells than DFO alone. Using ferrozine as an assay for iron measurement, levels of cellular iron in HepG2 cells treated with iron compounds correlated well with the extent of lipid peroxidation (r = 0.99 after log transformation). DP or DFO alone decreased levels of iron and lipid peroxidation in cells treated with iron. DFO + DP together had the most significant effect in preventing cells from lipid peroxidation but not as effective in decreasing overall iron levels in the cells. Using ESR spin trapping technique, we further tested factors that can affect oxidant-producing activity of Fe(2+) with dissolved O(2) in a cell-free system. Oxidant formation enhanced with increasing Fe(2+) concentrations and reached a maximum at 5 mM of Fe(2+). When the concentration of Fe(2+) was increased to 50 mM, the oxidant-producing activity of Fe(2+) sharply decreased to zero. The initial ratio of Fe(3+):Fe(2+) did not affect the oxidant producing activity of Fe(2+). However, an acidic pH (< 3.5) significantly slowed down the rate of the reaction. Our results suggest that reaction of Fe(2+) with O(2) is an important one for oxidant formation in biological system, and therefore, drugs capable of inhibiting redox activity of Fe(2+) should be considered in combination with a Fe(3+) chelator for iron overload chelation therapy.     

Effect of zinc on superoxide-dependent hydroxyl radical production in vitro

Trace elements play an important role in oxygen metabolism and therefore in the formation of free radicals. Whereas iron and copper are usually the main enhancers of free radical formation, other trace elements, such as zinc and selenium, protect against the harmful effects of these radicals. To investigate the different protective mechanisms of zinc on radical formation, we examined the effects of added zinc and copper on superoxide dismutase activity. We also studied the effects of copper and iron on xanthine oxidase activity and on the Haber-Weiss cycle (iron, superoxide, and hydrogen peroxide), which generates hydroxyl radicals in vitro. The hypoxanthine/xanthine oxidase radical generating system contained a variety of different physiological ligands for binding the iron. This study confirmed the inhibitory effect of copper on xanthine oxidase activity. Moreover, it demonstrated that zinc inhibited hydroxyl radical formation when this formation was catalyzed by a citrate-iron complex in the hypoxanthine/xanthine oxidase reaction. Finally, human blood plasma inhibited citrate-iron-dependent hydroxyl radical formation under the same conditions. Although trace elements seemed responsible for this antioxidant activity of plasma, it is likely that zinc played no role as a plasma antioxidant. Indeed, calcium appeared to be responsible for most of this effect under our experimental conditions.