Human cytosolic iron regulatory protein 1 contains a linear iron-sulfur cluster.
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Binding Sites↗
Biomedical subjects
G N George
Publications and source records attributed to G N George.
Fluorine encapsulation and stabilization of biologically relevant low-valence copper-oxo cores.
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Copper↗
Yeast Sco1, a protein essential for cytochrome c oxidase function is a Cu(I)-binding protein.
Sco1 is a conserved essential protein, which has been implicated in the delivery of copper to cytochrome c oxidase, the last enzyme of the electron transport chain. In this study, we show for the first time that the purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer. X-ray absorption spectroscopy suggests that the Cu(I) is ligated via three ligands, and we show that two cysteines, present in a conserved motif CXXXC, and a conserved histidine are involved in Cu(I) ligation. The mutation of any one of the conserved residues in Sco1 expressed in yeast abrogates the function of Sco1 resulting in a non-functional cytochrome c oxidase complex. Thus, the function of Sco1 correlates with Cu(I) binding. Data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo.
Amino Acid Motifs↗
Synthesis, characterization, and electrochemistry of cis-oxothio- and cis-bis(thio)tungsten(VI) complexes of hydrotris(3,5-dimethylpyrazol-1-yl)borate.
The complexes TpWO2X react with sulfiding agents such as B2S3 or P4S10 to give the oxothio- and bis(thio)tungsten(VI) complexes TpWOSX (X = Cl(-)) and TpWS2X [X = Cl(-), S2PPh2(-); Tp = hydrotris(3,5-dimethylpyrazol-1-yl)borate]. The reaction of TpWS2Cl with (i) PPh3 in pyridine and (ii) dimethyl sulfoxide affords TpWOSCl in good overall yield. The chloro complexes undergo metathesis with alkali metal salts to yield species of the type TpWOSX and TpWS2X [X = OPh(-), SPh(-), SePh(-), (-)-mentholate]. The diamagnetic complexes exhibit NMR spectra indicative of C(1) (TpWOSX) or C(s) (TpWS2X) symmetry and IR spectra consistent with terminal oxo and thio ligation (nu(W=O), 940-925 cm(-1); nu(W=S) or nu(WS2), 495-475 cm(-1)). Crystals of (R,S)-TpWOS[(-)-mentholate] are monoclinic, space group P2(1), with a = 11.983(2) A, b = 18.100(3) A, c = 13.859(3) A, beta = 91.60(2) degrees, V = 3004.6(8) A(3), and Z = 4. Crystals of TpWS2(OPh)-CH2Cl2 are orthorhombic, space group Pbca, with a = 16.961(4) A, b = 33.098(7) A, c = 9.555(2) A, V = 5364(2) A(3), and Z = 8. The mononuclear, distorted-octahedral tungsten centers are coordinated by a tridentate Tp ligand, an alkoxy or aryloxy ligand, and two terminal chalcogenide ligands. The average W=O and W=S distances are 1.726(7) and 2.125(2) A, respectively, and the O=W=S and S=W=S angles 102.9(3) and 102.9(1) degrees, respectively. The tungsten and sulfur X-ray absorption spectra of TpWOSCl and TpWS2Cl are consistent with the presence of terminal pi-bonded thio ligands in both complexes. The thio complexes generally undergo a reversible one-electron reduction at potentials significantly more positive than their oxo analogues. The chemical, spectroscopic, and electrochemical properties of the complexes are heavily influenced by the presence of W=S pi frontier orbitals.
Aldehyde Oxidoreductases↗
In situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi.
Sulfur K-edge X-ray absorption spectroscopy has been used to determine the chemical identity of the sulfur-containing species in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) in situ, before and after cell disruption. The major sulfur-containing species in the intact root is sinigrin (1-thio-beta-D-glucopyranose 1-N-(sulfoxy)-3-buteneimidate) and related congeners. Disrupting the cells by applying local pressure allowed the conversion of the sulfur moieties in sinigrin to isothiocyanates and sulfate in approximately equimolar amounts. In contrast to previous suggestions, no detectable thiocyanates were formed, but an unusual thio intermediate may have been identified for the first time.
Brassica↗
Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.
Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.
Chlorobi↗
The mitochondrial copper metallochaperone Cox17 exists as an oligomeric, polycopper complex.
Cox17 is the candidate copper metallochaperone for delivery of copper ions to the mitochondrion for assembly of cytochrome c oxidase. Cox17 purified as a recombinant molecule lacking any purification tag binds three Cu(I) ions per monomer in a polycopper cluster as shown by X-ray absorption spectroscopy. The CuCox17 complex exists in a dimer/tetramer equilibrium with a 20 microM k(d). The spectroscopic data do not discern whether the dimeric complex forms a single hexanuclear Cu(I) cluster or two separate trinuclear Cu(I) clusters. The Cu(I) cluster(s) exhibit(s) predominantly trigonal Cu(I) coordination. The cluster(s) in Cox17 resemble(s) the polycopper clusters in Ace1 and the Cup1 metallothionein in being pH-stable and luminescent. The physical properties of the CuCox17 complex purified as an untagged molecule differ from those reported previously for a GST-Cox17 fusion protein. The CuCox17 cluster is distinct from the polycopper cluster in Cup1 in being labile to ligand exchange. CuCox17 localized within the intermitochondrial membrane space appears to be predominantly tetrameric, whereas the cytosolic CuCox17 is primarily a dimeric species. Cys-->Ser substitutions at Cys23, Cys24, or Cys26 abolish the Cox17 function and prevent tetramerization, although Cu(I) binding is largely unaffected. Thus, the oligomeric state of Cox17 may be important to its physiological function.
Blotting, Western↗
Molybdenum sequestration in Brassica species. A role for anthocyanins?
To elucidate plant mechanisms involved in molybdenum (Mo) sequestration and tolerance, Brassica spp. seedlings were supplied with molybdate, and the effects on plant physiology, morphology, and biochemistry were analyzed. When supplied with (colorless) molybdate Indian mustard (Brassica juncea) seedlings accumulated water-soluble blue crystals in their peripheral cell layers. Energy dispersive x-ray analysis showed that Mo accumulated predominantly in the vacuoles of the epidermal cells. Therefore, the blue crystals are likely to be a Mo compound. The x-ray absorption spectrum of the plant-accumulated Mo was different than that for molybdate, indicating complexation with a plant molecule. Because the blue compound was water soluble and showed a pH-dependent color change, possible involvement of anthocyanins was investigated. An anthocyanin-less mutant of Brassica rapa ("fast plants") was compared with varieties containing normal or high anthocyanin levels. The anthocyanin-less mutant did not show accumulation of a blue compound when supplied with molybdate. In the anthocyanin-containing varieties, the blue compound colocalized with anthocyanins in the peripheral cell layers. Mo accumulation by the three B. rapa varieties was positively correlated with anthocyanin content. Addition of molybdate to purified B. rapa anthocyanin resulted in an in vitro color change from pink to blue. Therefore, Mo appears to be sequestered in vacuoles of the peripheral cell layers of Brassica spp. as a blue compound, probably a Mo-anthocyanin complex.
Adaptation, Physiological↗
Deep desulfurization of extensively hydrodesulfurized middle distillate oil by Rhodococcus sp. strain ECRD-1.
Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by Rhodococcus sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.
Biodegradation, Environmental↗
The active site structure of Thalassiosira weissflogii carbonic anhydrase 1.
X-ray absorption spectroscopy at the Zn K-edge indicates that the active site of the marine diatom Thalassiosira weissflogii carbonic anhydrase is strikingly similar to that of mammalian alpha-carbonic anhydrase enzymes. The zinc has three histidine ligands and a single water at 1.98 A. This is quite different from the beta-carbonic anhydrases of higher plants in which zinc is coordinated by two cysteine thiolates, one histidine, and a water molecule. The diatom carbonic anhydrase shows no significant sequence similarity with other carbonic anhydrases and may represent an example of convergent evolution at the molecular level.
Animals↗
Quantitative, chemically specific imaging of selenium transformation in plants.
Quantitative, chemically specific images of biological systems would be invaluable in unraveling the bioinorganic chemistry of biological tissues. Here we report the spatial distribution and chemical forms of selenium in Astragalus bisulcatus (two-grooved poison or milk vetch), a plant capable of accumulating up to 0.65% of its shoot dry biomass as Se in its natural habitat. By selectively tuning incident x-ray energies close to the Se K-absorption edge, we have collected quantitative, 100-microm-resolution images of the spatial distribution, concentration, and chemical form of Se in intact root and shoot tissues. To our knowledge, this is the first report of quantitative concentration-imaging of specific chemical forms. Plants exposed to 5 microM selenate for 28 days contained predominantly selenate in the mature leaf tissue at a concentration of 0.3-0.6 mM, whereas the young leaves and the roots contained organoselenium almost exclusively, indicating that the ability to biotransform selenate is either inducible or developmentally specific. While the concentration of organoselenium in the majority of the root tissue was much lower than that of the youngest leaves (0.2-0.3 compared with 3-4 mM), isolated areas on the extremities of the roots contained concentrations of organoselenium an order of magnitude greater than the rest of the root. These imaging results were corroborated by spatially resolved x-ray absorption near-edge spectra collected from selected 100 x 100 microm(2) regions of the same tissues.
Image Processing, Computer-Assisted↗
Stoichiometry of complex formation between Copper(I) and the N-terminal domain of the Menkes protein.
The inherent cellular toxicity of copper ions demands that their concentration be carefully controlled. The cellular location of the Menkes ATPase, a key element in the control of intracellular copper, is regulated by the intracellular copper concentration through the N-terminus of the enzyme, comprising 6 homologous subdomains or modules, each approximately 70 residues in length and containing a -Cys-X-X-Cys- motif. Based on the proposal that binding of copper to these modules regulates the Menkes ATPase cellular location by promoting changes in the tertiary structure of the enzyme, we have expressed the entire N-terminal domain (MNKr) and the second metal-binding module (MNKr2) of the Menkes protein in E. coli and purified them to homogeneity. Ultraviolet-visible, luminescence, and X-ray absorption spectroscopy show that copper and silver bind to the single module, MNKr2, with a stoichiometry of one metal ion per module. However, the array of six modules, MNKr, binds Cu(I) to produce a homogeneous conformer with 4 mol equiv of metal ion. The metal ions are bound in an environment that is shielded from solvent molecules. We suggest a model of the Menkes protein in which the Cu(I) binding induces tertiary changes in the organization of the six metal-binding domains.
Adenosine Triphosphatases↗
Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase.
Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A.
Amino Acid Sequence↗
Structural basis of the antagonism between inorganic mercury and selenium in mammals.
Mercuric chloride toxicity in mammals can be overcome by co-administration of sodium selenite. We report a study of the mutual detoxification product in rabbit plasma, and of a Hg-Se-S-containing species synthesized by addition of equimolar mercuric chloride and sodium selenite to aqueous, buffered glutathione. Chromatographic purification of this Hg-Se-S species and subsequent structural analysis by Se and Hg extended X-ray absorption fine structure (EXAFS) spectroscopy revealed the presence of four-coordinate Se and Hg entities separated by 2.61 A. Hg and Se near-edge X-ray absorption spectroscopy of erythrocytes, plasma, and bile of rabbits that had been injected with solutions of sodium selenite and mercuric chloride showed that Hg and Se in plasma samples exhibited X-ray absorption spectra that were essentially identical to those of the synthetic Hg-Se-S species. Thus, the molecular detoxification product of sodium selenite and mercuric chloride in rabbits exhibits similarities to the synthetic Hg-Se-S species. The underlying molecular mechanism for the formation of the Hg-Se-S species is discussed.
Animals↗
Reduction and coordination of arsenic in Indian mustard.
The bioaccumulation of arsenic by plants may provide a means of removing this element from contaminated soils and waters. However, to optimize this process it is important to understand the biological mechanisms involved. Using a combination of techniques, including x-ray absorption spectroscopy, we have established the biochemical fate of arsenic taken up by Indian mustard (Brassica juncea). After arsenate uptake by the roots, possibly via the phosphate transport mechanism, a small fraction is exported to the shoot via the xylem as the oxyanions arsenate and arsenite. Once in the shoot, the arsenic is stored as an As(III)-tris-thiolate complex. The majority of the arsenic remains in the roots as an As(III)-tris-thiolate complex, which is indistinguishable from that found in the shoots and from As(III)-tris-glutathione. The thiolate donors are thus probably either glutathione or phytochelatins. The addition of the dithiol arsenic chelator dimercaptosuccinate to the hydroponic culture medium caused a 5-fold-increased arsenic level in the leaves, although the total arsenic accumulation was only marginally increased. This suggests that the addition of dimercaptosuccinate to arsenic-contaminated soils may provide a way to promote arsenic bioaccumulation in plant shoots, a process that will be essential for the development of an efficient phytoremediation strategy for this element.
Arsenic↗
Fate of selenate and selenite metabolized by Rhodobacter sphaeroides.
Cultures of a purple nonsulfur bacterium, Rhodobacter sphaeroides, amended with approximately 1 or approximately 100 ppm selenate or selenite, were grown phototrophically to stationary phase. Analyses of culture headspace, separated cells, and filtered culture supernatant were carried out using gas chromatography, X-ray absorption spectroscopy, and inductively coupled plasma spectroscopy-mass spectrometry, respectively. While selenium-amended cultures showed much higher amounts of SeO(3)(2-) bioconversion than did analogous selenate experiments (94% uptake for SeO(3)(2-) as compared to 9.6% for SeO(4)(2-)-amended cultures from 100-ppm solutions), the chemical forms of selenium in the microbial cells were not very different except at exposure to high concentrations of selenite. Volatilization accounted for only a very small portion of the accumulated selenium; most was present in organic forms and the red elemental form.
Chromatography, Gas↗
Generation and biomimetic chemistry of tungsten-dithiolene complexes containing the hydrotris(3,5-dimethylpyrazol-1-yl)borate ligand.
Reactions of bis(thio)-W(VI) complexes, LWS2X (L = hydrotris (3,5-dimethylpyrazol-1-yl)borate, X = monoanion), with alkynes produce dithiolene complexes, LWX(dithiolene). The synthesis and characterization of orange LW(OPh){S2C2(CO2Me)2} (1) and burgundyred LW(SePh) {S2C2(Ph)(2-quinoxalinyl)} (2) and the X-ray crystal structure of 1.0.5CH2Cl2, are described in detail. Crystals of 1.0.5CH2Cl2 are orthorhombic, space group Pbcn, with a = 29.826(6), b = 13.291(4), c = 16.078(4) A, V = 6373(5) A3, and Z = 8. The six-coordinate, distorted-octahedral complex features a tridentate L ligand, a monodentate phenoxide ligand, and a bidentate dithiolene ligand. The short W-S bonds (2.267(4) and 2.279(4) A) and the parameters associated with the phenoxide ligand (W-O = 1.850(8) A, W-O-C = 146(1) degree), point to a considerable degree of W-ligand multiple bonding in the [W(OPh)(dithiolene)]+ unit. For 2, W-Se and average W-S distances of 2.49(2) A and 2.30(2) A, respectively, have been obtained from EXAFS studies. The formation of yellow 3,3'-dithiobis[2-(phenyl)thieno[2,3-b]quinoxaline] (3) upon oxidation of 2 supports the likely generation of urothione upon oxidative degradation of molybdopterin-containing tungsten enzymes from hyperthermophilic organisms.
Boric Acids↗
X-ray absorption spectroscopy of cadmium phytochelatin and model systems.
Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides. We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly. We have used the synthetic ions [Cd(SPh)4]2-, [Cd4(SPh)10]2- and [S4Cd10(SPh)16]4-as crystallographically defined models for the cadmium site. The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes. In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species. The Cd-S distance in the phytochelatin complex is 2.54 A. The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation. Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS. The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for [S4Cd10(SPh)16]4-. This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.
Cadmium↗