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PubMed2004

Ferroxidase activity in a laccase-like multicopper oxidase from Liriodendron tulipifera.

Ferroxidase activity was detected in a laccase-like multicopper oxidase (LMCO) produced in transgenic tobacco cells expressing an LMCO cDNA (Ltlacc2.2) cloned from yellow-poplar (Liriodendron tulipifera). This marks the first report of ferroxidase activity associated with a plant laccase and suggests that some members of this plant enzyme family may have physiological functions based on activities other than their more widely recognized phenoloxidase activity. Recent work with LMCOs from bacteria, yeast and mammals has shown that metal oxidase activities in these enzymes can be important for their primary physiological functions, With respect to ferroxidase activity in certain plant LMCOs, it is proposed that the high levels of LMCO expression in plant vascular tissues may reflect the need for high-efficiency iron uptake pumps in tissues that undergo lignification during normal development. Such iron uptake pumps would function to minimize levels of free iron so that reactive oxygen species do not reach toxic levels when H2O2 is generated for peroxidase-mediated monolignol coupling during lignin deposition.

Amino Acid Sequence↗

PubMed2005

Recombinant expression and functional characterization of human hephaestin: a multicopper oxidase with ferroxidase activity.

Human hephaestin (Hp) is a transmembrane protein that has been implicated in duodenal iron export. Mutations in the murine hephaestin gene (sla) produce microcytic, hypochromic anemia that is refractory to oral iron therapy. Hp shares approximately 50% sequence identity with the plasma multicopper ferroxidase ceruloplasmin including conservation of residues involved in disulfide bond formation and metal coordination. On the basis of this similarity to ceruloplasmin, human hephaestin may also bind copper and possess ferroxidase activity. To test this hypothesis, human hephaestin cDNA has been cloned by reverse transcription of human duodenal mRNA. Following in vitro mutagenesis to make the encoded polypeptide suitable for expression and purification, the hephaestin cDNA was cloned into the expression vector pNUT and introduced into baby hamster kidney cells. After selection with methotrexate, the baby hamster kidney cells secreted the recombinant human hephaestin into the medium at a level of approximately 2 mg/L. Purification was achieved by a single immunoaffinity chromatography step. As judged by SDS-PAGE, N-terminal sequence analysis, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, the purified hephaestin was homogeneous with a mass of 129600 Da, suggesting a carbohydrate content of 7.7%. Inductively coupled plasma mass spectrometry revealed that recombinant hephaestin contained an average of 3.13 atoms of copper per protein molecule. A visible absorption maximum was observed at 607 nm, consistent with the presence of a Type 1 copper site. By using ferrous ammonium sulfate as a substrate, recombinant hephaestin was shown to have ferroxidase activity with a K(m) of 2.1 microM for Fe(II). Lastly, urea PAGE showed that hephaestin was able to catalyze formation of diferric transferrin from Fe(II) and apotransferrin.

Animals↗

PubMed2004

Ferrous binding to the multicopper oxidases Saccharomyces cerevisiae Fet3p and human ceruloplasmin: contributions to ferroxidase activity.

The multicopper oxidases are a family of enzymes that couple the reduction of O(2) to H(2)O with the oxidation of a range of substrates. Saccharomyces cerevisiae Fet3p and human ceruloplasmin (hCp) are members of this family that exhibit ferroxidase activity. Their high specificity for Fe(II) has been attributed to the existence of a binding site for iron. In this study, mutations at the E185 and Y354 residues, which are putative ligands for iron in Fet3p, have been generated and characterized. The effects of these mutations on the electronic structure of the T1 Cu site have been assessed, and the reactivities of this site toward 1,4-hydroquinone (a weak binding substrate) and Fe(II) have been evaluated and interpreted in terms of the semiclassical Marcus theory for electron transfer. The electronic and geometric structure of the Fe(II) substrate bound to Fet3p and hCp has been studied for the first time, using variable-temperature variable field magnetic circular dichroism (VTVH MCD) spectroscopy. The iron binding sites in Fet3p and hCp appear to be very similar in nature, and their contributions to the ferroxidase activity of these proteins have been analyzed. It is found that these iron binding sites play a major role in tuning the reduction potential of iron to provide a large driving force for the ferroxidase reaction, while still supporting the delivery of the Fe(III) product to the acceptor protein. Finally, the analysis of possible electron-transfer (ET) pathways from the protein-bound Fe(II) to the T1 Cu site indicates that the E185 residue not only plays a role in iron binding, but also provides the dominant ET pathway to the T1 Cu site.

Amino Acid Substitution↗

PubMed2004

Kinetic analysis of bovine spleen apoferritin and recombinant H and L chain homopolymers: iron uptake suggests early stage H chain ferroxidase activity and second stage L chain cooperation.

Ferritin utilizes ferroxidase activity to incorporate iron. Iron uptake kinetics of bovine spleen apoferritin (H: L = 1 : 1.1) were compared with those of recombinant H chain ferritin and L chain ferritin homopolymers. H chain ferritin homopolymer showed an iron uptake rate identical to bovine spleen apoferritin (0.19 and 0.21 mmol/min/micromol of protein, respectively), and both showed iron concentration-dependent uptake. In contrast, the L chain homopolymer, which lacks ferroxidase, did not incorporate iron and showed the same level of iron autoxidation in the absence of ferritin. Bovine spleen apoferritin was shown to have two iron concentration-dependent uptake pathways over a range of 0.02-0.25 mM ferrous ammonium sulfate (FAS) by an Eadie-Scatchard plot (v/[FAS] versus v), whereas the H chain ferritin homopolymer was found to have only one pathway. Of the two Km values found in bovine spleen apoferritin, the lower mean Km value was 9.0 microM, while that of the H chain homopolymer was 11.0 microM. H chain ferritin homopolymer reached a saturating iron uptake rate at 0.1 mM FAS, while bovine spleen apoferritin incorporated more iron even at 0.25 mM FAS. These results suggest that the intrinsic ferroxidase of ferritin plays a significant role in iron uptake, and the L chain cooperates with the H chain to increase iron uptake.

Animals↗

PubMed1996

Evidence that the specificity of iron incorporation into homopolymers of human ferritin L- and H-chains is conferred by the nucleation and ferroxidase centres.

Mammalian ferritins are iron-storage proteins made of 24 subunits of two types: the H- and L-chains. L-chains, in contrast with H-chains, lack detectable ferroxidase activity. When ferritins were subjected to iron loading in vitro with increments near the saturation limit of 4000 Fe atoms per molecule, the homopolymers of human H-chains formed insoluble aggregates, caused by non-specific iron hydrolysis, whereas the homopolymers of L-chains remained soluble and incorporated most of the available iron. To analyse the molecular reasons for the difference, Glu-57 and Glu-60, which are conserved and exposed on the cavity of L-chains, were substituted with His, as in H-chains. The double substitution made the L-homopolymers as sensitive as the H-homopolymers to the iron-induced aggregation, whereas the opposite substitution in the H-chain increased homopolymer resistance to the aggregation only marginally. Millimolar concentrations of citrate and phosphate increased iron incorporation in H-homopolymers by reducing non-specific iron hydrolysis, but inhibited that in L-homopolymers by sequestering available iron. The data indicate that the specific iron incorporation into L-homopolymers is mainly due to the iron-nucleation capacity of Glu-57, Glu-60 and other carboxyl groups exposed on the cavity; in contrast, the specificity of iron incorporation into H-homopolymers is related to its ferroxidase activity, which determines rapid Fe(III) accumulation inside the cavity. The finding that ferroxidase centres are essential for the incorporation of iron in the presence of likely candidates of cellular iron transport, such as phosphate and citrate, confirms their importance in ferritin function in vivo.

Amino Acids↗

PubMed1999

Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism.

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

Cations↗

PubMed2002

The ferroxidase activity of yeast frataxin.

Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (alpha(48)) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (alpha --> alpha(3)). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O(2) is detected at Fe(II)/mYfh1p ratios of < or = 0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, alpha(3), is responsible for both reactions. The observed 2 Fe(II)/O(2) stoichiometry implies production of H(2)O(2) during the ferroxidase reaction. However, only a fraction of the expected total H(2)O(2) is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H(2)O(2) reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H(2)O(2) results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O(2). This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer.

Enzyme Activation↗

PubMed2002

Molecular basis of H2O2 resistance mediated by Streptococcal Dpr. Demonstration of the functional involvement of the putative ferroxidase center by site-directed mutagenesis in Streptococcus suis.

H(2)O(2) is an unavoidable cytotoxic by-product of aerobic life. Dpr, a recently discovered member of the Dps protein family, provides a means for catalase-negative bacteria to tolerate H(2)O(2). Potentially, Dpr could bind free intracellular iron and thus inhibit the Fenton chemistry-catalyzed formation of toxic hydroxyl radicals (H(2)O(2) + Fe(2+) --> (.)OH + (-)OH + Fe(3+)). We explored the in vivo function of Dpr in the catalase- and NADH peroxidase-negative pig and human pathogen Streptococcus suis. We show that: (i) a Dpr allelic exchange knockout mutant was hypersensitive ( approximately 10(6)-fold) to H(2)O(2), (ii) Dpr incorporated iron in vivo, (iii) a putative ferroxidase center was present in Dpr, (iv) single amino acid substitutions D74A or E78A to the putative ferroxidase center abolished the in vivo iron incorporation, and (v) the H(2)O(2) hypersensitive phenotype was complemented by wild-type Dpr or by a membrane-permeating iron chelator, but not by the site-mutated forms of Dpr. These results demonstrate that the putative ferroxidase center of Dpr is functionally active in iron incorporation and that the H(2)O(2) resistance is mediated by Dpr in vivo by its iron binding activity.

Amino Acid Sequence↗

PubMed2007

A multicopper ferroxidase involved in iron binding to transferrins in Dunaliella salina plasma membranes.

The halotolerant alga Dunaliella salina is unique among plants in that it utilizes a transferrin (TTf) to mediate iron acquisition (Fisher, M., Zamir, A., and Pick, U. (1998) J. Biol. Chem. 273, 17553-17558). Two new proteins that are induced by iron deprivation were identified in plasma membranes of D. salina as follows: a multicopper ferroxidase termed D-Fox and an internally duplicated glycoprotein (p130B). D-Fox and p130B are accessible to glycolytic, proteolytic, and biotin surface tagging treatments, suggesting that they are surface-exposed glycoproteins. Induction of D-Fox was also manifested by ferroxidase activity in plasma membrane preparations. These results are puzzling because ferroxidases in yeast and in Chlamydomonas reinhardtii function in redox-mediated iron uptake, a mechanism that is not known to operate in D. salina. Two lines of evidence suggest that D-Fox and p130B interact with D. salina triplicated transferrin (TTf). First, chemical cross-linking combined with mass spectroscopy analysis showed that D-Fox and p130B associate with TTf and with another plasma membrane transferrin. Second, detergent-solubilized D-Fox and p130B comigrated on blue native gels with plasma membrane transferrins. 59Fe autoradiography indicated that this complex binds Fe3+ ions. Also, the induction of D-Fox and p130B is kinetically correlated with enhanced iron binding and uptake activities. These results suggest that D-Fox and p130B associate with plasma membrane transferrins forming a complex that enhances iron binding and iron uptake. We propose that the function of D-Fox in D. salina has been modified during evolution from redox-mediated to transferrin-mediated iron uptake, following a gene transfer event of transferrins from an ancestral animal cell.

Algal Proteins↗

PubMed1985

Duodenal xanthine oxidase (EC 1.2.3.2) and ferroxidase activities in the rat in relation to the increased iron absorption caused by peroral xylitol.

The effect of peroral administration of xylitol on the absorption of iron and the activities of xanthine oxidase (EC 1.2.3.2) and ferroxidase in rat duodenal wall was studied. Adult male rats were given the basal diet containing 200 g xylitol/kg or the same diet containing no added carbohydrates for 8 weeks. Both feeding groups comprised twelve animals. Xylitol significantly increased serum and liver Fe concentrations with a concomitant, significant increase in the duodenal xanthine oxidase activities, but caused a marginal increase in the duodenal ferroxidase activities. In vitro, sugar alcohols reduced the binding rate of Fe to transferrin. The xylitol-induced increase of Fe absorption may involve the following mechanism: the high intraluminal xylitol concentration of the xylitol-fed rats keeps Fe in the form of a soluble complex for a prolonged period of time, due to the slow absorption of xylitol. The polyol-Fe complex in turn induces xanthine oxidase and ferroxidase formation.

Administration, Oral↗

PubMed1998

Role of serum ceruloplasmin and its ferroxidase activity on preterm labor.

The role of serum ceruloplasmin and its ferroxidase activity on preterm labor with or without rupture of the membranes was studied. Thirty-seven primigravid women between 31 and 35 weeks of gestation were investigated. Our material is divided into 3 groups. Group A consisted of 12 patients with premature rupture of the membranes; group B consisted of 10 patients with preterm labor but intact membranes, and group C consisted of 15 non-laboring patients. Patients with rupture of the membranes presented with statistically significantly higher serum ceruloplasmin levels compared to those with preterm labor but intact membranes (p < 0.05) as well as to non-laboring patients (p < 0.01). On the contrary, the mean ferroxidase activity level or ceruloplasmin was 518.2+/-183.42, 460.9+/-198.8 and 517.5+/-142.01 micromol/l/min in groups A, B and C, respectively. These values were not significantly different. Our results show a loss of ferroxidase activity of ceruloplasmin in patients with rupture of the membranes and probably suggest a defect in the maternal defense mechanisms.

Adult↗

PubMed1996

Effect of smoking on ceruloplasmin and its ferroxidase activity in pregnant women.

This study deals with the effects of smoking on serum ceruloplasmin and its ferroxidase activity in pregnant women. The mean value (+/- SD) of serum ceruloplasmin in women smokers during pregnancy is significantly higher (p < 0.01) in comparison to that of nonsmokers, whereas the ferroxidase activity of ceruloplasmin in smokers is not significantly different from that of nonsmokers. These findings suggest that ceruloplasmin in women smokers loses part of its ferroxidase activity.

Adult↗

PubMed1988

An automated method for the kinetic measurement of ferroxidase activity.

The oxidase activity of caeruloplasmin towards Fe II (ferroxidase) appears to be of physiological significance both in iron metabolism and as a serum anti-oxidant. We have developed an automated assay for ferroxidase using a centrifugal analyser. The method is quick and precise, and is not subject to interference by haemolysis, jaundice or lipaemia. Ferroxidase activities correlate significantly with caeruloplasmin and copper concentrations. Normal reference ranges have been measured.

Adult↗

PubMed1986

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.

Animals↗

PubMed1995

Identification of the ferroxidase centre of Escherichia coli bacterioferritin.

The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.

Amino Acid Sequence↗

PubMed1998

Automated measurement of serum ferroxidase activity.

A method is described for automated measurement of serum ceruloplasmin ferroxidase activity. In this method, Fe2+ ions are used as the substrate. In addition, a new calibration system without ceruloplasmin is also presented. Optimum assay reaction conditions were determined. Maximal catalytic activity was obtained at 0.45 mol/L acetate buffer, pH 5.8. The reagents and calibrator are stable for at least 6 months. Significant correlations between serum ferroxidase and p-phenylenediamine oxidase activities (r = 0.96; P <0.0001) and copper concentration (r = 0.93; P <0.0001) were found. The range for serum ceruloplasmin ferroxidase activity in healthy persons was 198-1107 U/L, and in patients with bronchial asthma it was 601-1912 U/L.

Asthma↗

PubMed1998

Iron loading into ferritin by an intracellular ferroxidase.

An intracellular, membrane-bound enzyme exhibiting both p-phenylenediamine oxidase activity and ferrous iron oxidase activity was isolated with the plasma membrane fraction of horse heart and studied for its ability to load iron into ferritin. The ferroxidase activity of the tissue oxidase was stimulated approximately twofold by horse spleen apoferritin, and the iron was loaded into ferritin. The loading of iron into ferritin by the tissue oxidase was inhibited by anti-horse serum ceruloplasmin antibody. The stoichiometry of iron oxidation and oxygen consumption during iron loading into ferritin by the tissue-derived oxidase and serum ceruloplasmin were 3.6 +/- 0.2 and 3.9 +/- 0.2, respectively. These data provide evidence that an enzyme analogous to ceruloplasmin is present on the plasma membrane of horse heart and that this ferroxidase is capable of catalyzing the loading of iron into ferritin. The implications of these data on the present models for the uptake and storage of iron by cells are discussed.

Animals↗

PubMed2003

Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from Saccharomyces cerevisiae.

The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe(2+) to Fe(3+); this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe(2+), one of which is essential also to the coupling of the ferroxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K(M) for Fe(2+) exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in (59)Fe uptake. For the Fet3p(E185D) mutant, K(M) for iron was 300-fold greater than the wild-type K(M), while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe(2+ )binding to Fet3p and to the subsequent trafficking of the Fe(3+) produced to Ftr1p.

Binding Sites↗