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Journal of the American Chemical Society 2009 Dec; 131(50)
Unnatural amino Acid substitution as a probe of the allosteric coupling pathway in a mycobacterial cu(i) sensor.
The Cu(I) sensor Mycobacterium tuberculosis CsoR is a founding member of a new metalloregulatory protein family. Here we show that two "atom" substitutions of the Nepsilon2 face of a Cu(I) coordinating histisine-61 allosterically uncouple Cu(I) and D... expand abstractNA binding, with no effect on Cu(I) binding affinity and coordination structure. A model analogous to the allosteric switch mechanism in Staphylococcus aureus CzrA, a zinc sensor protein with a completely different fold, is proposed. collapse abstract
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Biochemistry 2009 Oct; 48(40)
Biophysical characterization of the iron in mitochondria from Atm1p-depleted Saccharomyces cerevisiae.
Atm1p is an ABC transporter localized in the mitochondrial inner membrane; it functions to export an unknown species into the cytosol and is involved in cellular iron metabolism. Depletion or deletion of Atm1p causes Fe accumulation in mitochondria a... expand abstractnd a defect in cytosolic Fe/S cluster assembly but reportedly not a defect in mitochondrial Fe/S cluster assembly. In this study the nature of the accumulated Fe was examined using Mossbauer spectroscopy, EPR, electronic absorption spectroscopy, X-ray absorption spectroscopy, and electron microscopy. The Fe that accumulated in aerobically grown cells was in the form of iron(III) phosphate nanoparticles similar to that which accumulates in yeast frataxin Yfh1p-deleted or yeast ferredoxin Yah1p-depleted cells. Relative to WT mitochondria, Fe/S cluster and heme levels in Atm1p-depleted mitochondria from aerobic cells were significantly diminished. Atm1p depletion also caused a buildup of nonheme Fe(II) ions in the mitochondria and an increase in oxidative damage. Atm1p-depleted mitochondria isolated from anaerobically grown cells exhibited WT levels of Fe/S clusters and hemes, and they did not hyperaccumulate Fe. Atm1p-depleted cells lacked Leu1p activity, regardless of whether they were grown aerobically or anaerobically. These results indicate that Atm1p does not participate in mitochondrial Fe/S cluster assembly and that the species exported by Atm1p is required for cytosolic Fe/S cluster assembly. The Fe/S cluster defect and the Fe-accumulation phenotype, resulting from the depletion of Atm1p in aerobic cells (but not in anaerobic cells), may be secondary effects that are observed only when cells are exposed to oxygen during growth. Reactive oxygen species generated under these conditions might degrade iron-sulfur clusters and lower heme levels in the organelle. collapse abstract
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Biochemistry 2009 Aug; 48(33)
Molecular structure and biochemical properties of the HCCH-Zn2+ site in HIV-1 Vif.
Virion infectivity factor (Vif) is an HIV accessory protein that is essential for the infection of CD4(+) T cells. Vif recruits a Cullin 5 (Cul5)-based ubiquitin ligase that targets a host cytidine deaminase, apolipoprotein B mRNA editing enzyme cata... expand abstractlytic polypeptide-like 3G (APOBEC3G), for proteasomal degradation. The Vif N-terminus binds APOBEC3G, and the C-terminus interacts with the Cul5-based ubiquitin ligase machinery. Within the C-terminus, a highly conserved H(108)-X(5)-C(114)-X(17-18)-C(133)-X(3-5)-H(139) (HCCH) motif binds zinc and is implicated in the Vif-Cul5 interaction. We have employed the biomimetic peptide HCCHp (HIV-1 Vif amino acids 101-142) in order to determine the zinc ligands and investigate the role of zinc binding in Cul5 recognition. Using CD spectroscopy, a competitive zinc binding assay, and a light scattering assay, we found that mutation of the conserved His and Cys residues in HCCHp had little effect on secondary structure but reduced zinc binding affinity and altered the aggregation properties of the peptides. X-ray absorption spectroscopy was used to study zinc coordination in wild-type HCCHp. The data are consistent with S(2)N(imid)(2) coordination and strongly suggest that His-108, Cys-114, Cys-133, and His-139 are zinc ligands. Mutation of one or both conserved Cys residues in HCCHp led to a decrease in Cys ligation, and an increase in the number of (N, O) ligands, with noninteger coordination numbers suggesting zinc site heterogeneity. A purified fragment of human Cul5 was found to inhibit zinc-induced aggregation of HCCHp, and pull-down experiments revealed that zinc binding to HCCHp increases the strength of the HCCHp-Cul5 interaction by 8-fold. collapse abstract
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Journal of structural biology 2009 May; 166(3)
Br-rich tips of calcified crab claws are less hard but more fracture resistant: a comparison of mineralized and heavy-element biological materials.
We find that the spoon-like tips of the chelipeds (large claws) of the crab Pachygrapsus crassipes differ from the rest of the claw in that they are not calcified, but instead contain about 1% bromine--thus they represent a new example of a class of ... expand abstractstructural biological materials that contain heavy elements such as Zn, Mn, Fe, Cu, and Br bound in an organic matrix. X-ray absorption spectroscopy data suggest that the bromine is bound to phenyl rings, possibly in tyrosine. We measure a broad array of mechanical properties of a heavy-element biological material for the first time (abrasion resistance, coefficient of kinetic friction, energy of fracture, hardness, modulus of elasticity and dynamic mechanical properties), and we make a direct comparison with a mineralized tissue. Our results suggest that the greatest advantage of bromine-rich cuticle over calcified cuticle is resistance to fracture (the energy of fracture is about an order of magnitude greater than for calcified cuticle). The greatest advantage relative to unenriched cuticle, represented by ant mandible cuticle, is a factor of about 1.5 greater hardness and modulus of elasticity.The spoon-like tips gain additional fracture resistance from the orientation of the constituent laminae and from the viscoelasticity of the material. We suggest that fracture resistance is of greater importance in smaller organisms, and we speculate that one function of heavy elements in structural biological materials is to reduce molecular resonant frequencies and thereby increase absorption of energy from impacts. collapse abstract
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Biochemistry 2009 Apr; 48(15)
Molecular insights into the metal selectivity of the copper(I)-sensing repressor CsoR from Bacillus subtilis.
Bacillus subtilis CsoR (Bsu CsoR) is a copper-sensing transcriptional repressor that regulates the expression of the copZA operon encoding a copper chaperone and a Cu efflux P-type ATPase, respectively. Bsu CsoR is a homologue of Mycobacterium tuberc... expand abstractulosis CsoR (Mtb CsoR), representative of a large Cu(I)-sensing regulatory protein family. We show here that Bsu CsoR binds approximately 1 mol equiv of Cu(I) per monomer in vitro with an affinity >or=10(21) M(-1). X-ray absorption spectroscopy shows Cu(I) adopts a trigonal S(2)N coordination like Mtb CsoR. Both apo and Cu(I)-bound Bsu CsoR are stable tetramers in the low micromolar monomer concentration range by sedimentation velocity and equilibrium ultracentrifugation. Apo-Bsu CsoR binds to a pseudopalindromic 30 bp copZA operator-promoter DNA with a stoichiometry of two tetramers per DNA and stepwise affinities of K(1)(apo) = 3.1(+/-0.8) x 10(7) M(-1) and K(2)(apo) = 8.3 (+/-2.2) x 10(7) M(-1) (0.4 M NaCl, 25 degrees C, pH 6.5). Cu(I) Bsu CsoR binds to the same DNA with greatly reduced affinities, K(1)(Cu) = 2.9(+/-0.4) x 10(6) M(-1) and K(2)(Cu)
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Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 2009 Apr; 14(4)
Auranofin disrupts selenium metabolism in Clostridium difficile by forming a stable Au-Se adduct.
Clostridium difficile is a nosocomial pathogen whose incidence and importance are on the rise. Previous work in our laboratory characterized the central role of selenoenzyme-dependent Stickland reactions in C. difficile metabolism. In this work we ha... expand abstractve identified, using mass spectrometry, a stable complex formed upon reaction of auranofin (a gold-containing drug) with selenide in vitro. X-ray absorption spectroscopy supports the structure that we proposed on the basis of mass-spectrometric data. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium ((75)Se) in selenoproteins in both Escherichia coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth medium significantly reduces the inhibitory action of auranofin on the growth of C. difficile. On the basis of these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. This study demonstrates that targeting selenium metabolism provides a new avenue for antimicrobial development against C. difficile and other selenium-dependent pathogens. collapse abstract
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Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 2009 Feb; 14(3)
The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a 4Fe-4S cluster.
In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CX(n)CCGX(m)CXXC), designated as the CCG domain i... expand abstractn the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Fe-2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), (57)Fe electron-nuclear double resonance (ENDOR) and Mössbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Fe-4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Fe-4S](2+) cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with g (zyx) = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with g (zyx) = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S(3)(O/N)(1) coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the iron-sulfur cluster and the zinc site. collapse abstract
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Molecular microbiology 2008 Dec; 71(2)
SurR: a transcriptional activator and repressor controlling hydrogen and elemental sulphur metabolism in Pyrococcus furiosus.
This work describes the identification and characterization of SurR, Pyrococcus furiosus sulphur (S(0)) response regulator. SurR was captured from cell extract using promoter DNA of a hydrogenase operon that is downregulated in the primary response o... expand abstractf P. furiosus to S(0), as revealed by DNA microarray experiments. SurR was validated as a sequence-specific DNA binding protein, and characterization of the SurR DNA binding motif GTTn(3)AAC led to the identification of several target genes that contain an extended motif in their promoters. A number of these were validated to contain upstream SurR binding sites. These SurR targets strongly correspond with open reading frames and operons both up- and downregulated in the primary response to S(0). In vitro transcription revealed that SurR is an activator for its own gene as well as for two hydrogenase operons whose expression is downregulated during the primary S(0) response; it is also a repressor for two genes upregulated during the primary S(0) response, one of which encodes the primary S(0)-reducing enzyme NAD(P)H sulphur reductase. Herein we give evidence for the role of SurR in both mediating the primary response to S(0) and controlling hydrogen production in P. furiosus. collapse abstract
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Biochemistry 2008 Oct; 47(40)
A Cu(I)-sensing ArsR family metal sensor protein with a relaxed metal selectivity profile.
ArsR (or ArsR/SmtB) family metalloregulatory homodimeric repressors collectively respond to a wide range of metal ion inducers in regulating homeostasis and resistance of essential and nonessential metal ions in bacteria. BxmR from the cyanobacterium... expand abstract Osciliatoria brevis is the first characterized ArsR protein that senses both Cu (I)/Ag (I) and divalent metals Zn (II)/Cd (II) in cells by regulating the expression of a P-type ATPase efflux pump (Bxa1) and an intracellular metallothionein (BmtA). We show here that both pairs of predicted alpha3N and alpha5 sites bind metal ions, but with distinct physicochemical and functional metal specificities. Inactivation of the thiophilic alpha3N site via mutation (C77S) abolishes regulation by both Cd (II) and Cu (I), while Zn (II) remains a potent allosteric negative effector of operator/promoter binding (Delta G c >or= +3.2 kcal mol (-1)). In contrast, alpha5 site mutant retains regulation by all four metal ions, albeit with a smaller coupling free energy (Delta G c approximately +1.7 (+/-0.1) kcal mol (-1)). Unlike the other metals ions, the BxmR dimer binds 4 mol equiv of Cu (I) to form an alpha3N binuclear Cu (I) 2S 4 cluster by X-ray absorption spectroscopy. BxmR is thus distinguishable from other closely related ArsR family sensors, in having evolved a metalloregulatory alpha3N site that can adopt an expanded range of coordination chemistries while maintaining redundancy in the response to Zn (II). The evolutionary implications of these findings for the ArsR metal sensor family are discussed. collapse abstract
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Biochemistry 2007 Nov; 46(44)
A cysteine-rich CCG domain contains a novel 4Fe-4S cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.
Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanis... expand abstracttic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site. collapse abstract
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Biochemistry 2007 Oct; 46(39)
Multiple metal binding domains enhance the Zn(II) selectivity of the divalent metal ion transporter AztA.
Transition metal-transporting P1B-type CPx ATPases play crucial roles in mediating metal homeostasis and resistance in all cells. The degree to which N-terminal metal binding domains (MBDs) confer metal specificity to the transporter is unclear. We s... expand abstracthow that the two MBDs of the Zn/Cd/Pb effluxing pump Anabaena AztA are functionally nonequivalent, but only with respect to zinc resistance. Inactivation of the a-MBD largely abrogates resistance to high intracellular Zn(II) levels, whereas inactivation of the b-MBD is not as deleterious. In contrast, inactivation of either the a- or b-MBD has little measurable impact on Cd(II) and Pb(II) resistance. The membrane proximal b-MBD binds Zn(II) with a higher affinity than the distal N-terminal a-MBD. Facile Zn(II)-specific intermolecular transfer from the a-MBD to the higher-affinity b-MBD is readily observed by 1H-15N HSQC spectroscopy. Unlike Zn(II), Cd(II) and Pb(II) form saturated 1:1 S4 or S3(O/N) complexes with AztAaHbH, where a single metal ion bridges the two MBDs. We propose that the tandem MBDs enhance Zn(II)-specific transport, while stabilizing a non-native inter-MBD Cd/Pb cross-linked structure that is a poor substrate and/or regulator for the transporter. collapse abstract
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Proceedings of the National Academy of Sciences of the United States of America 2007 Aug; 104(33)
Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity.
Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defi... expand abstractned. We have previously used Zn(2+) to modulate the assembly kinetics and morphology of congeners of the amyloid beta peptide (Abeta) associated with Alzheimer's disease. We now reveal a correlation among Abeta-Cu(2+) coordination, peptide self-assembly, and neuronal viability. By using the central segment of Abeta, HHQKLVFFA or Abeta(13-21), which contains residues H13 and H14 implicated in Abeta-metal ion binding, we show that Cu(2+) forms complexes with Abeta(13-21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Abeta(13-21)H14A, alters metal coordination, allowing Cu(2+) to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Abeta can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Abeta self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases. collapse abstract
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Journal of the American Chemical Society 2006 Mar; 128(11)
Modulating amyloid self-assembly and fibril morphology with Zn(II).
Metal ions (Zn(II)) are demonstrated as probes of amyloid structure in simple segments of the Abeta peptide, Abeta(13-21). By restricting the possible metal binding sites to His13/His14 dyad, we show that Zn2+ can specifically control the rate of sel... expand abstractf-assembly and dramatically regulate amyloid morphology via distinct coordination environments as characterized by X-ray absorption spectroscopy. The data establish that the single His13 is sufficient to coordinate Zn2+ productively for typical amyloid fiber formation, while a distinct Zn2+ coordination environment can be accessed in the presence of His13/Hi14 dyad to stabilize sheet/sheet associations and the transition to a ribbon/tube morphology. collapse abstract
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Journal of molecular biology 2006 Feb; 356(3)
Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA.
In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta ... expand abstractfold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase. collapse abstract
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FEBS letters 2005 Mar; 579(7)
Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase.
Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of micro... expand abstractbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S=1/2 [4Fe-4S]3) cluster, the EPR spectrum of which is broadened in the presence of CoM-33SH [Duin, E.C., Madadi-Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263-268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme. FEBS Lett. 538, 81-84]. These results provide indirect evidence that the disulfide binds to the iron-sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X-ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM-SeH). Se K edge extended X-ray absorption fine structure confirms a direct interaction of the Se in CoM-SeH-treated HDR with an Fe atom of the Fe-S cluster at an Fe-Se distance of 2.4A. collapse abstract
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The Journal of biological chemistry 2005 Mar; 280(10)
Rational design of a mononuclear metal site into the archaeal Rieske-type protein scaffold.
Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolo... expand abstractbus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds. collapse abstract
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Journal of synchrotron radiation 2004 Dec; 12(Pt 1)
Bottlenecks and roadblocks in high-throughput XAS for structural genomics.
Structural and functional characterization of the entire protein complement (the proteome) of an organism can provide an infrastructure upon which questions about biological pathways and systems biology can be framed. The technology necessary to perf... expand abstractorm this proteome-level structural and functional characterization is under development in numerous structural genomics and functional genomics initiatives. Given the ubiquity of metal active sites in a proteome, it seems appropriate to ask whether comprehensive local structural characterization of metal sites within a proteome (metalloproteomics) is either a valid or obtainable goal. With a proteome-wide knowledge of the active-site structures of all metalloproteins, one could start to ask how metal insertion, cluster assembly and metalloprotein expression are affected by growth conditions or developmental status etc. High-throughput X-ray absorption spectroscopy (HTXAS) is being developed as a technology for investigating the metalloproteome. In creating a pipeline from genome to metalloproteome, several bottlenecks to high-throughput determination of metal-site structures must be overcome. For example, automation of arraying small samples for XAS examination must be invented, automation of rapid data collection of multiple low-volume low-concentration samples must be developed, automation of data reduction and analysis must be perfected. Discussed here are the promises and the pitfalls of HTXAS development, including the results of initial feasibility experiments. collapse abstract
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Biochemistry 2004 Jul; 43(26)
A stable mercury-containing complex of the organomercurial lyase MerB: catalysis, product release, and direct transfer to MerA.
Bacteria isolated from organic mercury-contaminated sites have developed a system of two enzymes that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. Both enzymes are encoded on the mer... expand abstract operon and require sulfhydryl-bound substrates. The first enzyme is an organomercurial lyase (MerB), and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon-mercury bond, resulting in the formation of a reduced carbon compound and inorganic ionic mercury. Of several mercury-containing MerB complexes that we attempted to prepare, the most stable was a complex consisting of the organomercurial lyase (MerB), a mercuric ion, and a molecule of the MerB inhibitor dithiothreitol (DTT). Nuclear magnetic resonance (NMR) spectroscopy and extended X-ray absorption fine structure spectroscopy of the MerB/Hg/DTT complex have shown that the ligands to the mercuric ion in the complex consist of both sulfurs from the DTT molecule and one cysteine ligand, C96, from the protein. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, has been demonstrated by NMR and dialysis. We used an enzyme buffering test to determine that the MerB/Hg/DTT complex acts as a substrate for the mercuric reductase MerA. The observed MerA activity is higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA. This suggests that the mercuric ion can be transferred between the two enzymes by a direct transfer mechanism. collapse abstract
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Biochemistry 2004 Jul; 43(26)
Spectroscopic determination of the thermodynamics of cobalt and zinc binding to GATA proteins.
Vertebrate GATA proteins regulate processes that are vital to development, and each possesses two tandem GATA finger domains: an N-terminal GATA finger and a C-terminal GATA finger. These GATA fingers require Zn(2+) to fold, to bind DNA recognition e... expand abstractlements, and to regulate transcription. While the GATA-1 C-terminal finger is necessary and sufficient to bind to single GATA DNA sites, the N-terminal finger interacts with DNA such that the double finger unit (DF domain) has a binding and transactivation profile that is tuned by the DNA-binding site. Co(2+) was used as a spectroscopic probe in a series of competition titrations to determine the affinity of Co(2+) and Zn(2+) for the C-terminal finger from chicken GATA-1 and the double finger from human GATA-1 (referred to in this report as CF and DF). For CF, these experiments yielded K(b)(Co) = 1.0 (+/-1.3) x 10(7) M(-1) and K(b)(Zn) = 2.0 (+/-1.3) x 10(10) M(-1). For DF, these experiments yielded equilibrium constants for the process of two M(2+) binding to form M(2+)(2)-DF of beta(2)(Co) = 2.5 (+/-1.6) x 10(14) M(-2) and beta(2)(Zn) = 6.3 (+/-2.5) x 10(20) M(-2). The ZnS(4) coordination environment of Zn(2+)-bound CF was confirmed with X-ray absorption spectroscopy. A detailed analysis of these data suggests that the N-terminal and C-terminal fingers of DF act as independent and identical Zn(2+)-binding sites and each finger binds Zn(2+) with an affinity equivalent to that of CF. collapse abstract
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Journal of bacteriology 2004 Feb; 186(6)
Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site.
The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR reside... expand abstracts at the homodimer interface in a novel antiparallel arrangement of alpha-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue alpha-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more (203)Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step. collapse abstract
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The Journal of biological chemistry 2004 Mar; 279(13)
Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type 2Fe-2S ferredoxin from Sulfolobus solfataricus.
We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on th... expand abstracte properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation. collapse abstract
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Biochemistry 2003 Dec; 42(50)
X-ray absorption spectroscopic analysis of reductive 2Fe-2S cluster degradation in hyperthermophilic archaeal succinate:caldariellaquinone oxidoreductase subunits.
The biological [2Fe-2S] clusters play important roles in electron transfer and cellular signaling for a variety of organisms from archaea, bacteria to eukarya. The two recombinant hyperthermophilic archaeal [2Fe-2S] cluster-binding proteins, SdhC and... expand abstract the N-terminal domain fragment of SdhB, of Sulfolobus tokodaii respiratory complex II overproduced in Escherichia coli are thermostable as isolated, but moderately sensitive to reduction with excess dithionite. We used iron K-edge X-ray absorption spectroscopy to monitor the structural changes of their Fe sites in the irreversible [2Fe-2S] cluster degradation process. Regardless of the differences in the cluster-ligating cysteine motifs and the XAS-detectable [2Fe-2S](2+) cluster environments, a complete reductive breakdown of the [2Fe-2S] clusters resulted in the appearance of a new Fourier transform (FT) peak at approximately 3.3 A with a concomitant loss of the Fe-Fe interaction at ca. 2.7 A for both proteins. On the basis of the unambiguous assignment of the 3.3 A FT peak, our results suggest that a biological [2Fe-2S] cluster breakdown under reducing conditions generally releases Fe(2+) from the polypeptide chain into the aqueous solution, and the Fe(2+) might then be recruited as a secondary ferrous iron source for de novo biosynthesis and/or regulation of iron-binding enzymes in the cellular system. collapse abstract
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Journal of the American Chemical Society 2003 Dec; 125(48)
The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae is a dinuclear metallohydrolase.
The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.... expand abstracte. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 A, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 A, respectively. A second-shell feature at ca. 3.34 A appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, 2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented. collapse abstract
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The Journal of biological chemistry 2004 Jan; 279(4)
Transcription factor B contacts promoter DNA near the transcription start site of the archaeal transcription initiation complex.
Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life... expand abstract, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions -40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection. collapse abstract
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Protein science : a publication of the Protein Society 2003 Jun; 12(7)
Structural studies of the interaction of S-adenosylmethionine with the 4Fe-4S clusters in biotin synthase and pyruvate formate-lyase activating enzyme.
The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to genera... expand abstractte an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate. collapse abstract
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