Characterization of Escherichia coli NrdH. A glutaredoxin-like protein with a thioredoxin-like activity profile

Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases. Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes. Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase. Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis. The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH. Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function. Overexpression of E. coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase. Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione. We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site. We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.     

RT1-U: identification of a novel, active, class Ib alloantigen of the rat MHC

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.     

A Microscopic study of the transformation of sphalerite particles during the roasting of zinc concentrate

The formation of zinc ferrite (ZnFe₂O₄) during the roasting of iron-bearing zinc concentrates requires substantial additional processing to recover the zinc from this compound by leaching and to eliminate the iron from the leachate. The phase changes that occur in the particles of a typical industrial zinc sulfide concentrate during roasting in a fluidized bed at 1223 K were investigated by the use of light microscopy, electron microprobe analysis, and SEM with EDS. The processes which the iron undergoes during its eventual transformation into ferrite have been clarified by examination of the phases and the morphology of partially roasted marmatitic sphalerite particles (Zn, Fe)S, and by reference to the known phase equilibria involved in the Zn-Fe-S-0 system. The oxidation of ironbearing sphalerite occurs in three stages. The first involves the selective diffusion of most of the iron to the particle surface resulting in the formation of an iron oxide shell enclosing a largely unreacted zinc sulfide kernel. In the second stage, this kernel is oxidized to form a solid solution of zinc oxide and iron oxide. The iron is initially present in the ferrous state but, with the disappearance of the sulfide kernel, is oxidized to ferric iron. In the final stage, this dissolved iron oxide and the iron oxide shell react with the surrounding zinc oxide to form the refractory spinel zinc ferrite.     

The tyrosyl free radical of recombinant ribonucleotide reductase from Mycobacterium tuberculosis is located in a rigid hydrophobic pocket

The tyrosyl free radical in protein R2-2 of class Ib ribonucleotide reductase (RNR) fromMycobacterium tuberculosis is essential for the enzymatic activity and has an EPR spectrum remarkably similar to that of the tyrosyl radical YD* in PSII. The EPR relaxation properties of the radical suggest a very weak exchange coupling between the two redox centers, the radical and the diferric cluster. The tyrosyl radical gives almost identical EPR spectra in the temperature interval 10-293 K. We conclude that the tyrosyl radical sits in a rigid pocket. Two ring protons and one beta-methylene proton account for the major anisotropic hyperfine interactions. A high-frequency EPR spectrum of the radical showed a resolved gx = 2. 0092, indicating that a hydrogen bond to the phenolic oxygen of the radical is absent. Theoretical modeling studies based on the structural data known for Salmonella typhimurium class Ib RNR protein R2F revealed a hydrophobic wall aligned with the radical harboring residue Y110. The distance between the phenolic oxygen of the radical and the diferric cluster is longer in the two class Ib nrdF R2 proteins than in other characterized class Ia R2 proteins. The tyrosyl radical in protein R2-2 from M. tuberculosis was accessible to direct reduction by dithionite in the absence of a mediator. The radical could be partly regenerated when the system was exposed to O2 after the completion of anaerobic reduction. This indicates that the Fe3+ ions also had become reduced by dithionite.     

Resonance Raman study of sirohydrochlorin and siroheme in sulfite reductases from sulfate reducing bacteria

Soret-excited resonance Raman (RR) spectra are reported for the sirohemes in the oxidized and Cr11(EDTA)-reduced forms of both desulforubidin from D. baculatus (DSR) and the low molecular weight sulfite reductase from D. vulgaris (1SIR) and for sirohydrochlorin in the oxidized form of desulfoviridin from D. gigas (DSV). Several patterns in the RR spectra of these enzymes can be utilized as signatures for the siroheme/sirohydrochlorin moiety. The active site for DSR and 1SIR consists of a siroheme exchange-coupled to a [4Fe-4S]2+ cluster. Upon addition of Cr11(EDTA), the active center of DSR and 1SIR undergoes a one-electron and two-electron reduction, respectively. The RR spectra of DSR suggest that the siroheme iron is high spin and 5-coordinate in the oxidized enzyme and probably remains high spin and 5-coordinate upon reduction. The iron in the siroheme of oxidized 1SIR changes from a low spin and probably 6-coordinate configuration to a high spin, 5-coordinate complex upon two-electron reduction of the active site. Close similarities between the RR spectral features of the two-electron-reduced assimilatory sulfite reductases from E. coli and from D. vulgaris (1SIR) are discussed.     

Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa

Nitrite reductase (NiR) from Pseudomonas aeruginosa (EC 1.9.3.2) (NiR-Pa) is a soluble enzyme catalyzing the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which each monomer carries one c and one d1 heme. The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously called Thiosphaera pantotropha) (NiR-Pd) have been described [Fülop, V., et al. (1995) Cell 81, 369-377; Williams, P. A., et al. (1997) Nature 389, 406-412], and we recently reported on the structure of oxidized NiR-Pa at 2.15 A [Nurizzo, D., et al. (1997) Structure 5, 1157-1171]. Although the domains carrying the d1 heme are almost identical in both NiR-Pa and NiR-Pd oxidized and reduced structures, the c heme domains show a different pattern of c heme coordination, depending on the species and the redox state. The sixth d1 heme ligand in oxidized NiR-Pd was found to be Tyr25, whereas in NiR-Pa, the homologuous Tyr10 does not interact directly with Fe3+, but via a hydroxide ion. Furthermore, upon reduction, the axial ligand of the c heme of NiR-Pd changes from His17 to Met108. Finally, in the oxidized NiR-Pa structure, the N-terminal stretch of residues (1-29) of one monomer interacts with the other monomer (domain swapping), which does not occur in NiR-Pd. Here the structure of reduced NiR-Pa is described both in the unbound form and with the physiological product, NO, bound at the d1 heme active site. Although both structures are similar to that of reduced NiR-Pd, significant differences with respect to oxidized NiR-Pd were observed in two regions: (i) a loop in the c heme domain (residues 56-62) is shifted 6 A away and (ii) the hydroxide ion, which is the sixth coordination ligand of the heme, is removed upon reduction and NO binding and the Tyr10 side chain rotates away from the position adopted in the oxidized form. The conformational changes observed in NiR-Pa as the result of reduction are less extensive than those occurring in NiR-Pd. Starting with oxidized structures that differ in many respects, the two enzymes converge, yielding reduced conformations which are very similar to each other, which indicates that the conformational changes involved in catalysis are considerably diverse.     

Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp. N-771. Evidence of a novel post-translational modification in the cysteine ligand

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.     

Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.     

Exploration of the structural environment of the iron-sulfur cluster in putidaredoxin by nitrogen-15 NMR spectroscopy of selectively labeled cysteine residues

Putidaredoxin is a di-iron protein whose paramagnetic region is not well characterized by 1H detected NMR. We have studied the structure of this region in greater detail by directly observed 15N NMR of oxidized and reduced putidaredoxin preparations in which the six cysteine residues are selectively labeled with 15N. A new method for preparation of a stable form of reduced putidaredoxin has been developed for use in NMR. The 15N NMR spectra of the oxidized and reduced forms are characteristically different, and we have measured and compared 15N chemical shifts, spin-lattice relaxation times (T1), and chemical shift/temperature dependences for both forms. Evidence for localized valencies of the iron atoms in the reduced form is presented. From the 15N T1 values of the oxidized form, reduced distances of the cysteine backbone 15N nuclei from the center of the Fe2S2 cluster have been calculated. These distances are consistent with those calculated from X-ray crystal structure data for five ferredoxins, and confirm the structural similarity of the Fe2S2 clusters in putidaredoxin and in these ferredoxins in the oxidized state.     

磷酸-重铬酸钾分光光度法测定氧化锌酸性浸出液中的锰量

建立了分光光度法测定氧化锌酸性浸出液中锰量的新方法。试样在硫酸介质中用双氧水将二价铁离子氧化成三价铁离子,消除了亚铁离子的干扰,使重铬酸钾能在8~10 mol/L磷酸溶液中,室温下将Mn（Ⅱ）迅速定量地氧化为Mn（Ⅲ）,形成紫红色的锰（Ⅲ）-磷酸络和物,于分光光度计波长550 nm处测量其吸光度。样品测定具有较好的精密度,相对标准偏差为2.52%~3.81%,加标回收率为96.5%~104.6%。适合测定的范围为1.00~12.00 mg/mL。     

Expression of the leptin receptor in human leukaemic blast cells

The highly conserved residue F208 in protein R2 of E. coli ribonucleotide reductase is close to the binuclear iron center, and found to be involved in stabilizing the tyrosyl radical Y122. in wild type R2. Upon the reconstitution reaction of the mutant R2 F208Y with ferrous iron and molecular oxygen, we observed a new EPR singlet signal (g = 2.003) formed concomitantly with decay of the transient tyrosyl radical Y122. (g = 2.005). This new paramagnetic species (denoted Z) was stable for weeks at 4 degrees C and visible by EPR only below 50 K. The EPR singlet could not be saturated by available microwave power, suggesting that Z may be a mainly metal centered species. The maximum amount of the compound Z in the protein purified from cells grown in rich medium was about 0.18 unpaired spin/R2. An identical EPR signal of Z was found also in the double mutant R2 F208Y/Y122F. In the presence of high concentration of sodium ascorbate, the amounts of both the transient Y122. and the new species Z increased considerably in the reconstitution reaction. The results suggest that Z is most likely an oxo-ferryl species possibly in equilibrium with a Y208 ligand radical.     

电位滴定法测定铁矿石中氧化亚铁

铁矿石中氧化亚铁在检测过程中易被氧化,而采用电位滴定法测定时可直接在密闭容器中插入电极进行检测,从而避免亚铁离子被氧化,因此试验建立了电位滴定法测定铁矿石中氧化亚铁的方法。采用盐酸溶解样品,氟化铵加入量为0.5g使硅酸盐充分分解,样品分解时间为15min,无需加入硫磷混酸。以电位滴定法代替滴定法进行检测,对仪器工作条件进行了正交试验优化,设定滴定终点筛选标准(EPC)为20,最小加液量为20μL,信号漂移速率为50mV/min。实验方法用于测定4个铁矿石及岩石类标准样品中氧化亚铁,结果的相对标准偏差(RSD,n=10)不大于0.40%,测定值与认定值基本一致,相对误差(RE)不大于0.33%;分别按照实验方法和国标方法GB/T 6730.8—2016测定9个铁矿石及岩石类标准样品中氧化亚铁,测定值与国标方法测定值相吻合。     

FhuF, an iron-regulated protein of Escherichia coli with a new type of [2Fe-2S] center

We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K-12. The FhuF protein was labeled with a His-tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and M?ssbauer and EPR spectroscopy, FhuF was identified as a [2Fe-2S] protein. The g values (gx = 1.886, gy = 1.961, gz = 1.994) and some of the M?ssbauer parameters of FhuF obtained [oxidized protein as isolated: delta EQ,4.2K = 0.474 mm s-1; Fe3+ (reduced protein): delta EQ = 0.978 mm s-1] are not typical of common [2Fe-2S] proteins and indicate that FhuF has unusual structural properties. The primary sequence of FhuF does not show any sequence similarities to known [2Fe-2S] proteins. By site-directed mutagenesis, each of the six cysteines of FhuF was replaced by serine. EPR of the six reduced mutant proteins revealed that the terminal cysteine residues 244, 245, 256, and 259 form the [2Fe-2S]Cys4 cluster. Mutants having the Cys-to-Ser replacement at positions 244, 245, 256, or 259 did not complement a fhuF mutant. The motif Cys-Cys-Xaa10-Cys-Xaa2-Cys in FhuF differs considerably from the motif Cys-Xaa2-Cys-Xaa9-15-Cys-Xaa2-Cys found in other [2Fe-2S] proteins. The unusual Cys-Cys terminal group of the cluster may explain the atypical EPR and M?ssbauer spectroscopic properties of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is distorted. The phenotype of fhuF mutants and the structural features of the FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B.     

锌窑渣氧化气固相反应过程分析

基于锌窑渣氧化气固相氧化反应的脱硫曲线,分析了氧化脱硫过程有明显差异的温度范围。对锌窑渣中Cu、Zn及Fe的物相组成在不同温度范围内的变化进行了研究,并据此讨论了氧化过程中Cu、Zn及Fe的行为和反应机制。结果表明：大部分的硫化锌及硫化亚铁在398K-1073K的温度范围内将被氧化,而较多的硫化亚铜是在1073K-1373K的温度范围内被氧化。铜、锌氧化产物均易与铁氧化产物化合为铁酸盐。氧化过程中形成的铁酸锌不利于锌的挥发,仅当温度大于1073K时锌才会挥发,此时铁酸锌含量已达到高温分解与化合平衡含量。     

溴-甲醇非水体系分离-重铬酸钾滴定法测定直接还原铁中金属铁

金属铁含量是直接还原铁质量的主要指标,而直接还原铁是优质钢生产不可缺少的原料。因此测定直接还原铁中金属铁含量对优化钢材结构和提高钢的质量具有重要意义。采用溴-甲醇非水体系溶解直接还原铁中金属铁(MFe),使用聚四氟乙烯(PTFE)微孔过滤膜(孔径0.45 μm)抽滤分离残渣,金属铁以三溴化铁的形式存在于滤液中,从而实现了其与其他价态铁的分离。滤液中加入硫酸和多次加入过氧化氢,加热冒硫酸烟去除溴化物和甲醇以避免其干扰后续金属铁的测定。用氯化亚锡还原滤液中大部分三价铁为二价铁,以钨酸钠为指示剂,三氯化钛还原滤液中剩余的三价铁,用稀重铬酸钾溶液氧化过剩的三氯化钛。以二苯胺磺酸钠为指示剂,用重铬酸钾标准溶液滴定二价铁的含量。据此,建立了溴-甲醇非水体系分离-重铬酸钾滴定法测定直接还原铁中金属铁的方法。选取5个直接还原铁样品,按照实验方法进行精密度试验,测定结果的相对标准偏差(RSD,n=9)为0.24%～0.54%;将实验方法应用于2个直接还原铁标准样品中金属铁的测定,测得结果与认定值的相对误差为0.012%～0.15%。     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm

Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of Escherichia coli. One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase. Thioredoxin 1 is kept in a reduced state by thioredoxin reductase. In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation in vivo. In the Escherichia coli periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD. In a dsbD null mutant, DsbC accumulates in the oxidized form. This oxidized form is then able to promote disulfide bond formation. In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves. Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the E. coli periplasm. In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a dsbA strain defective for disulfide bond formation. Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant. We conclude that the in vivo redox function of thioredoxin 1 depends on the redox environment in which it is localized.     

Behavior of direct reduced iron and hot briquetted iron in the upper blast furnace shaft: Part I. Fundamentals of kinetics and mechanism of oxidation

It is considered that the use of prereduced ferrous materials and sources of metallic iron such as direct reduced iron (DRI) or hot briquetted iron (HBI) improves the productivity of the blast furnace (BF). However, oxidation of DRI/HBI can occur in the upper zone of the BF, which may increase the content of the reducing gases but may not decrease the coke rate substantially. The behavior of DRI and HBI was investigated by measuring the rate of oxidation of the materials in CO₂ gas in a temperature range of 400 °C to 900 °C. In addition, the microstructure of “as-received” and oxidized materials was examined. The iron oxide phases formed due to oxidation were determined using X-ray diffraction (XRD) and a vibrating sample magnetometer. The results of isothermal experiments indicated that the kinetics of oxidation of metallic iron is slow at 400 °C. In DRI samples, the initial rate is controlled by the limited mixed control of chemical kinetics at the iron/iron oxide interface and pore mass transfer, whereas gas diffusion in pores is the rate governing step during the final stages of oxidation. The oxidation of wustite from iron is found to be faster than the oxidation of the former to magnetite. The structure of DRI after oxidation resembled a “reverse topochemical-oxide on the surface metal in the center” structure at 600 °C to 700 °C. The final iron oxide phase formed in DRI after oxidation was magnetite and not hematite. The oxidation of HBI was limited to the surface of the samples at lower temperatures; at 900 °C, moderate oxidation was observed and a topochemical iron oxide layer was formed.     

The next generation of research: views of a sibling-psychiatrist-researcher

Ceruloplasmin purified from horse serum was rapidly reduced upon addition of increasing equivalents of ferrous iron, generating an electronically and conformationally distinct form. This form of ceruloplasmin was characterized by significant (80%) loss of EPR detectable type I and type II copper(II), complete loss of visible absorbance at 610 nm, as well as decreased hydrophobic surface area. The reduced form of ceruloplasmin slowly reduced molecular oxygen to complete its catalytic cycle. The presence of varied concentrations of apoferritin, but not apotransferrin, significantly enhanced the rate of ceruloplasmin oxidation. The magnitude of this stimulatory effect increased as the molar ratio of ceruloplasmin to apoferritin approached 1.0, shown previously to be the optimum ratio for loading iron into ferritin. The rate of ferrous iron oxidation by ceruloplasmin was significantly stimulated by the presence of apoferritin; however, apotransferrin had no effect. The length of time required for ceruloplasmin to oxidize all the iron and return to the native form of the enzyme was also affected by the concentration of iron. In addition, the rate of iron loading into ferritin was dependent upon ferrous iron concentration. These results provide evidence for the formation of a specific complex between the reduced form of ceruloplasmin and apoferritin and that reduction of ceruloplasmin by ferrous iron may be the signal for complex formation.     

Fluorescent pseudomonad pyoverdines bind and oxidize ferrous ion

Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe2+ -pyoverdine than for Fe3+ -pyoverdine. At pH 7.4, about 90% of Fe3+ was bound by pyoverdine Pa-C after 24 h whereas Fe2+ was bound by the pyoverdine completely in only 5 min. The possibility that Fe2+ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe2+ concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe2+ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe3+ -specific chelating agent, resulted in the formation of a Fe3+ -hydroxyquinoline complex, suggesting that the iron in the Fe2+ -pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.