A purified ferredoxin from Giardia duodenalis

A ferredoxin has been purified to homogeneity from the ancient protozoan parasite Giardia duodenalis. As far as we know, this is the first electron transport protein to be characterised from the organism. The ferredoxin exhibits absorption maxima at 296 and 406 nm with molar absorption coefficients of epsilon 296 = 16,650 +/- 240 M-1 cm-1 and epsilon 406 = 13,100 +/- 370 M-1 cm-1 respectively. The A406/A296 ratio ranged over 0.78-0.82. The molecular mass of the apoprotein calculated by mass spectrometry was 5730 +/- 100Da and the minimum molecular mass by amino acid analysis was 5926Da. There were four cysteine residues/molecule protein but no methionine, arginine, histidine or tyrosine. The absence of these latter residues is consistent with the amino acid content of most ferredoxins. The N-terminal amino acid sequence exhibited greatest similarity to Desulfovibrio gigas ferredoxin II and indicated the potential to coordinate an iron-sulfur cluster. There were 3.21 +/- 0.41 mol sulfide and 2.65 +/- 0.06 mol iron/mol protein. Electron paramagnetic resonance studies of this protein have indicated the presence of an iron-sulfur centre consistent with those of known ferredoxins. Ferredoxin serves as a biological electron acceptor from giardial pyruvate dehydrogenase with metronidazole as a terminal electron acceptor. Such a pathway may serve as a possible mechanism for the reductive activation of metronidazole in this parasite. A second ferredoxin has been purified to homogeneity, but at this stage there is insufficient material to fully characterise this protein. No other low-molecular-mass electron transport proteins have been identified in Giardia under the growth conditions described.     

Bicarbonate binding to the non-heme iron of photosystem II investigated by Fourier transform infrared difference spectroscopy and 13C-labeled bicarbonate

The binding site of the non-heme iron of photosystem II (PS II) is investigated by light-induced Fourier tranform infrared (FTIR) difference spectroscopy on Tris-washed membranes. The non-heme iron is oxidized (Fe3+) in the dark with ferricyanide and reduced (Fe2+) after light-induced charge separation by electron transfer from the semiquinone anion QA-. EPR experiments and IR modes of ferri- and ferrocyanide show that the electron donor side of PS II is reduced in less than 2 s after a flash and that ferricyanide reoxidizes the non-heme iron with a half-time of approximately 20 s. Recording FTIR spectra before and 2 s after flash illumination thus results in the Fe2+/Fe3+ difference spectrum. This spectrum shows band shifts and intensity changes of IR modes from ligands and neighboring residues of the non-heme iron. The IR modes of bicarbonate are revealed by comparison of Fe2+/Fe3+ spectra obtained on PS II membranes with 12C or 13C isotope labeled bicarbonate in H2O and in 2H2O. The nu as(CO) and nu s(CO) modes of bicarbonate in the Fe2+ state are assigned at 1530 +/- 10 and 1338 cm-1, respectively. The low frequency of the nu as(CO) mode is taken as experimental evidence that bicarbonate is a ligand of the non-heme iron. Furthermore, the small frequency difference (192 cm-1) between the nu as(CO) and nu s(CO) modes as compared to even hydrogen-bonded ionic bicarbonate strongly indicates that bicarbonate is a bidentate ligand of the non-heme iron in PS II. Upon iron oxidation, the bicarbonate modes are largely affected. The nu s(CO) mode is assigned at 1228 cm-1, while the nu as(CO) mode is tentatively assigned at 1658 +/- 20 cm-1. The strong up- and downshifts of the nu as and nu s(CO) modes of bicarbonate upon iron oxidation results in a frequency difference of 430 +/- 20 cm-1 that is not only explained by the increased charge on the iron but indicates that bicarbonate is a monodentate ligand of the oxidized iron. The sensitivity of the nu s(CO) mode of bicarbonate to 1H/2H exchange in both the Fe2+ and Fe3+ states and the presence in the Fe2+ state of a delta (COH) mode at 1258 cm-1 confirm that bicarbonate and not carbonate is the iron ligand and further exhibits hydrogen bond(s) with the protein. The 13C isotope-sensitive modes of bicarbonate are not affected by 15N labeling of the PS II membranes. 15N sensitive signals at 1111/1102 and 1094 cm-1 are assigned to side chain modes from histidine ligands of the iron. The latter signal is proposed to account for a histidine ligand that deprotonates upon iron oxidation. The involvement of protein peptide groups and side chains in the hydrogen-bond network around the iron is also discussed.     

A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport

The ATX1 gene of Saccharomyces cerevisiae was originally identified as a multi-copy suppressor of oxidative damage in yeast lacking superoxide dismutase. We now provide evidence that Atx1p helps deliver copper to the copper requiring oxidase Fet3p involved in iron uptake. atx1Delta null mutants are iron-deficient and are defective in the high affinity uptake of iron. These defects due to ATX1 inactivation are rescued by copper treatment, and the same has been reported for strains lacking either the cell surface copper transporter, Ctr1p, or the putative copper transporter in the secretory pathway, Ccc2p. Atx1p localizes to the cytosol, and our studies indicate that it functions as a carrier for copper that delivers the metal from the cell surface Ctr1p to Ccc2p and then to Fet3p within the secretory pathway. The iron deficiency of atx1 mutants is augmented by mutations in END3 blocking endocytosis, suggesting that a parallel pathway for intracellular copper trafficking is mediated by endocytosis. As additional evidence for the role of Atx1p in iron metabolism, we find that the gene is induced by the same iron-sensing trans-activator, Aft1p, that regulates CCC2 and FET3.     

Structural investigations on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine alpha-amidating enzyme

The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The enzyme contains 2 coppers per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized enzyme indicates that 98 +/- 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2-3 histidines and 1-2 O/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced enzyme data indicates that the nonhistidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands together with 0.5 S/CI ligand per copper. The value of v(CO) (2093 cm-1) derived from FTIR spectroscopy suggests coordination of a weak donor such as methionine, which is supported by a previous observation that the delta Pro-PHM382s mutant M314I is totally inactive. Binding of the peptide substrate N-Ac-Tyr-Val-Gly causes minimum structural perturbation at the Cu(I) centers but appears to induce a more rigid conformation in the vicinity of the S-Met ligand. The unusually intense 8983 eV Cu K-absorption edge feature in reduced and substrate-bound-reduced enzymes is suggestive of a trigonal or digonal coordination environment for Cu(I). A structural model is proposed for the copper centers involving 3 histidines as ligands to CuIA and 2 histidines and 1 methionine as ligands to CuIB. However, in view of the intense 8934 eV edge feature and the lack of CO-binding ability, a 2-coordinate structure for CuA is also entirely consistent with the data.     

Resonance Raman spectra of iron(III)-, copper(II)-, cobalt(III)-, and manganese(III)-transferrins and of bis(2,4,6-trichlorophenolato)diimidazolecopper(II) monohydrate, a possible model for copper(II) binding to transferrins

Fe(III), Cu(II), Co(III), and Mn(III) complexes of ovo- and human serum transferrins show resonance enhanced Raman bands near 1600, 1500, 1270, and 1170 cm-1 upon excitation with laser frequencies which fall within the visible absorption bands of those metalloproteins. Comparison of the visible absorption and resonance Raman spectra of the Cu(II)-transferrin complexes with those for the Cu(II) model compound, bis(2,4,6-trichlorophenolato)diimidazolecopper(II) monohydrate, indicates that the resonance Raman bands are due to enhancement of phenolic vibrational modes. For the model (Cu(II) compound, a normal coordinate analysis was used to aid our assignment of the observed resonance bands at 1562, 1463, 1311, and 1122 cm-1 to A1 vibrational modes of the 2,4,6-trichlorophenolato moiety. These assignments are consistent with those made for Cu(II)-transferrins. The latter assignments were based upon calculated A1 frequencies for p-methylphenol (Cummings, D.L., and Wood, J.L. (1974), J. Mol. Struct. 20, 1). The wavelength shifts in the resonance bands for the model compound from those for Cu(II)-transferrins are due to the influence of the chloro substituents on the planar vibrations of phenol. These results clearly identify tyrosine as a ligand in copper binding to transferrins.     

HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense

HAH1 is a 68-amino acid protein originally identified as a human homologue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 delta yeast. Molecular modeling of HAH1 predicts a protein structure of two alpha-helices overlaying a four-stranded antiparallel beta-sheet with a potential metal binding site involving two conserved cysteine residues. Consistent with this model, in vitro studies with recombinant HAH1 directly demonstrated binding of Cu(I), and site-directed mutagenesis identified these cysteine residues as copper ligands. Expression of wild type and mutant HAH1 in atx1 delta yeast revealed the essential role of these cysteine residues in copper trafficking to the secretory compartment in vivo, as expression of a Cys-12/Cys-15 double mutant abrogated copper incorporation into the multicopper oxidase Fet3p. In contrast, mutation of the highly conserved lysine residues in the carboxyl terminus of HAH1 had no effect on copper trafficking to the secretory pathway but eliminated the antioxidant function of HAH1 in sod1 delta yeast. Taken together, these data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biological processes in eucaryotic cells.     

Characterization of Euphorbia characias latex amine oxidase

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 x 10(5) M-1 cm-1 and 6000 M-1 cm-1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.     

Removal of iron and formation of copper(i) from solutions containing iron(ii) and copper(ii) by addition of chloride ion or acetonitrile

Solutions of Cu(II) and Fe(II) establish the redox equilibrium

$\text{Cu(II) + Fe(II)}\text{?}\text{K}\text{Cu(I) + Fe(III)}$

which is displaced to the right by addition of either Cl^? or acetonitrile (AN). Log K varies from ?10.5 in water to about ?2.5 in 4 M NaCl or AN, allowing iron to be removed selectively from copper (II) solutions either by solvent extraction with Versatic acid or by precipitation as goethite or j jarosite. To establish the required conditions Eh-pH diagrams have been developed for the CuH₂OCl and CuH₂OANSO₄²-systems at 25°C and 90°C. It is demonstrated that the catalytic effect of Cu(II) on the oxidation of Fe(II) to Fe(III) by O₂ is dependent on the concentration of Cl^? or AN and on the position of this redox equilibrium. Applications to removing iron from hydrometallurgical solutions are discussed and tested.     

冰铜中全铁的测定

重铬酸钾容量法测定矿石及其它原料中全铁的含量是一种普遍采用的方法，但是对于铜含量高的特殊样品一如测定冰铜中的含铁量，在用SnCl2还原三价铁的同时也将部分二价铜还原为一价铜，因此在用重铬酸钾标准溶液滴定的过程中一价铜也参与反应并促使二价铁的氧化，使滴定终点不明显且托延，因而影响测定结果，所以必须采取相应的措施——预分离，然后用重铬酸钾滴定铁，否则将无法得到正确结果。     

The effect of Fe(II) and Fe(III) on the efficiency of copper electrowinning from dilute acid Cu(II) sulphate solutions with the chemelec cell. Part II. The efficiency of copper electrowinning from dilute liquors

This programme of work forms part of a study into the application of the Chemelec cell for direct electrowinning of copper from dilute leach liquors. A typical liquor is defined as containing less than 2 g l^?1 Cu(II), with an equivalent or greater concentration of iron as Fe(II) and Fe(III). Electrowinning experiments using simulated liquors showed that the current efficiency decreased in proportion to the increase in the Fe(III) concentration. A current efficiency of less than 30% was obtained during the electrowinning of a copper solution (1–2 g l^?1 Cu) with an Fe(III) concentration of 0.5–3.0 g l^?1. Electrowinning from leach liquors (1–2 g l^?1 Cu, 1–2 g l^?1 Fe(III) and 1–5 g l^?1 Fe(II)) showed improved efficiencies. A current efficiency of 47% was obtained for copper removal to less than 50 mg l^?1, with an electrolytic energy cost of about £198 per tonne at 3.3p per kWh. The impurity content of the copper was low, the most significant impurity being lead at 0.012%. This work has shown that the Chemelec cell can achieve reasonable efficiencies for direct electrowinning from dilute leach liquors. Further work is required in order to confirm the efficiency of electrowinning and to assess the operating and capital costs of a commercial cell.     

Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway

The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.     

Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: an unexpected role for intracellular chloride channels

GEF1 is a gene in Saccharomyces cerevisiae, which encodes a putative voltage-regulated chloride channel. gef1 mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p. The defect is due to an inability to transfer Cu+ to apoFet3p within the secretory apparatus. We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl-. Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl- there is very little Cu-loading at any pH. Cl- has a positive allosteric effect on Cu-loading of apoFet3p. Kinetic studies suggest that Cl- may also bind to Fet3p and that Cu+ has an allosteric effect on the binding of Cl- to the enzyme. Thus, Cl- may be required for the metal loading of proteins within the secretory apparatus. These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.     

Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins

UV-visible spectroscopy, electrode oximetry, and pH stat were used to study Fe(II) oxidation and hydrolysis in horse spleen ferritin (HoSF) and recombinant human H-chain and L-chain ferritins (HuHF and HuLF). Appropriate test reactions and electrode responses were measured, establishing the reliability of oxygen electrode/pH stat for kinetics studies of iron uptake by ferritin. Stoichiometric ratios, Fe(II)/O2 and H+/Fe(II), and rates of oxygen uptake and proton production were simultaneously measured as a function of iron loading of the protein. The data show a clear distinction between the diiron ferroxidase site and mineral surface catalyzed oxidation of Fe(II). The oxidation/hydrolysis reaction attributed to the ferroxidase site has been determined for the first time and is given by 2Fe2+ + O2 + 3H2O --> [Fe2O(OH)2]2+ + H2O2 + 2H+ where [Fe2O(OH)2]2+ represents the hydrolyzed dinuclear iron(III) center postulated to be a mu-oxo-bridged species from UV spectrometric titration data and absorption band maxima. The transfer of iron from the ferroxidase site to the mineral core has been now established to be [Fe2O(OH)2]2+ + H2O --> 2FeOOH(core) + 2H+. Regeneration of protein ferroxidase activity with time is observed for both HoSF and HuHF, consistent with their having enzymatic properties, and is facilitated by higher pH (7.0) and temperature (37 degreesC) and by the presence of L-subunit and is complete within 10 min. In accord with previous studies, the mineral surface reaction is given by 4Fe2+ + O2 + 6H2O --> 4FeOOH(core) + 8H+. As the protein progressively acquires iron, oxidation/hydrolysis increasingly shifts from a ferroxidase site to a mineral surface based mechanism, decreasing the production of H2O2.     

Tumor cell cytotoxicity of a novel metal chelator

We have synthesized a novel six-coordinate metal chelator from the triamine cis-1,3,5-triaminocyclohexane by the addition of a 2-pyridylmethyl pendant arm on each nitrogen, which we term tachpyr. The experiments described here were designed to explore whether this compound exhibits potential antitumor activity. When added to MBT2 or T24 cultured bladder cancer cells, tachpyr was profoundly cytotoxic, with an IC50 of approximately 4.6 micromol/L compared with 70 micromol/L for desferioxamine. To explore the mode of action of tachpyr, several metal complexes were prepared, including Fe(II), Ca(II), Mn(II), Mg(II), Cu(II), and Zn(II) tachpyr complexes. Of these, the Zn(II), Cu(II), and Fe(II) complexes were without toxic effect, whereas the Ca(II), Mn(II), and Mg(II) complexes remained cytotoxic. To further probe the role of Zn(II) and Cu(II) chelation in the cytotoxicity of tachpyr, sterically hindered tachpyr derivatives were prepared through N-alkylation of tachpyr. These derivatives were unable to strongly bind Fe(III) or Fe(II) but were able to bind Zn(II) and Cu(II). When added to cells, these sterically hindered tachpyr derivatives were nontoxic, consistent with a role of iron depletion in the cytotoxic mechanism of tachpyr. Further, the addition of tachpyr to proliferating cultures resulted in an early and selective inhibition of ferritin synthesis, an iron storage protein whose translation is critically dependent on intracellular iron pools. Taken together, these experiments suggest that tachpyr is a cytotoxic metal chelator that targets intracellular iron, and that the use of tachpyr in cancer therapy deserves further exploration.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

Cementation of copper from complex sulphide leach liquor

As an alternative hydrometallurgical route, the complex sulphide concentrates (Zn, 18%; Cu, 3.5% and Pb, 9%) from Ambamata were leached in FeCl₃ solution. The resultant leach solution contained Zn, 7.3; Cu, 0.54; Pb, 1.35 g/l, besides iron (of which 67% was Fe(II) state). In order to recover copper from the leach liquor, cementation with mild steel, iron turnings, electroplated nickel, aluminium and lead substrates was studied. With iron turnings, more than 95% of the copper from the leach solution could be recovered at room temperature, pH 2.06 and air flow rate of 33–50 ml/s. Cementation could also be done on mild steel and aluminium substrates. The liquor after cementation contained lead and zinc as well as iron (mostly as Fe(II)) and is therefore suitable for further solvent extraction of lead and zinc.     

高铁生物堆浸液Na2S2O3回收铜新工艺

在生物浸铜过程中,Fe3+杂质逐步积累,当堆浸液中Fe3+浓度过高时,将使铜的萃取难以进行.本研究采用硫代硫酸钠处理高铁生物堆浸液使铜得到回收,当高铁生物堆浸液Cu2+为7.41g/L、TFe为27.9 g/L、Fe3+为14.68 g/L时,在反应温度为80℃,硫代硫酸钠用量为18 g/L,反应时间为60 min条件...     

EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the type 2 copper centers of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015)

EPR and 1H, 14,15N ENDOR spectra are described for the type 1 and type 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans. The study was carried out on preparations of NiR containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers. This has enabled ENDOR studies of type 1 and type 2 sites to be carried out largely independently of each other, by appropriate choice of the excitation field. Spectra were recorded both in the absence and presence of nitrite, allowing a clear determination of which of the two types of Cu center constitutes the substrate binding site. The EPR results show large changes in the type 2 site gparallel (which decreases by 0.065) and CuAparallel (which increases by 2.0 mT) while the type 1 site EPR is not affected. In addition, both 1H and 14N ENDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected. Our results clearly demonstrate that nitrite binds to the type 2 copper and that this process significantly perturbs the ligation of this copper by the protein histidine residues. No 15N ENDOR resonances were observed from 15N nitrite. The accessibility of the copper sites to solvent has been studied using 2H2O. The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bound to the copper atom.