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Michael W W Adams - One of the best experts on this subject based on the ideXlab platform.
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Nuclear resonance vibrational spectroscopy (NRVS) of Rubredoxin and MoFe protein crystals
Hyperfine Interactions, 2013Co-Authors: Eric Brecht, Michael W W Adams, Yuming Xiao, Kristen Aznavour, Hongxin Wang, Simon J. George, Stephen Keable, John W. Peters, Francis E. Jenney Jr., Wolfgang SturhahnAbstract:We have applied ^57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Iron-sulfur protein crystals, including oxidized wild type Rubredoxin crystals from Pyrococcus furiosus , and the MoFe protein of nitrogenase from Azotobacter vinelandii . Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented Rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type Rubredoxin crystals from Pyrococcus furiosus . The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the crystal structure.
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rubrerythrin from the hyperthermophilic archaeon pyrococcus furiosus is a Rubredoxin dependent iron containing peroxidase
Journal of Bacteriology, 2004Co-Authors: Michael V Weinberg, Francis E Jenney, Michael W W AdamsAbstract:Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 ± 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85°C using hydrogen peroxide with reduced P. furiosus Rubredoxin as the electron donor. The specific activity was 36 μmol of Rubredoxin oxidized/min/mg with apparent Km values of 35 and 70 μM for hydrogen peroxide and Rubredoxin, respectively. When rubrerythrin was combined with Rubredoxin and P. furiosus NADH:Rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 μmol of H2O2 reduced/min/mg of rubrerythrin at 85°C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide:Rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.
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A neutron crystallographic analysis of a Rubredoxin mutant
Applied Physics A, 2002Co-Authors: T. Chatake, Francis E Jenney, Michael W W Adams, Kazuo Kurihara, Ichiro Tanaka, Irina Tsyba, Nobuo NiimuraAbstract:In order to study the unusual thermostability of Rubredoxin from Pyrococcus furiosus (RubPf), the structure of a ‘triple mutant’ of this Rubredoxin, which is less thermostable than the wild type, was solved at 1.5-A resolution by neutron-diffraction analysis using the BIX-3 diffractometer at the JRR-3M reactor of JAERI. The positions of the non-hydrogen atoms are almost the same as the native Rubredoxin; however, for Trp3 → Tyr3, large structural changes were found, so that their hydrogen-bonding schemes are significantly different. Some positions of the hydrogen atoms and molecules of hydration are shifted in certain regions, suggesting that such differences may contribute to the differences in thermostability between this ‘triple-mutant’ Rubredoxin and wild-type Rubredoxin.
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5 Rubredoxin from pyrococcus furiosus
Methods in Enzymology, 2001Co-Authors: Francis E Jenney, Michael W W AdamsAbstract:Publisher Summary Rubredoxins are small, redox-active proteins that range in size from 45 to 55 amino acids. More than a dozen Rubredoxins have been purified and characterized and the protein and complete genome databases contain 26 Rubredoxinlike sequences. So far, Rubredoxin has been found exclusively in strict anaerobes, which includes both bacteria and archaea, and some organisms that might be considered microaerophiles. There are a number of much larger proteins that contain Rubredoxin-like sequences or domains and are found in facultative anaerobes and aerobes. Such proteins vary considerably in size of, as well as location of and distance between, the cysteine motifs. The canonical Rubredoxin has been purified from only one hyperthermophile, the archaeon Pyrococcus furiosus . This chapter describes the purification of Rubredoxin from P. furiosus and of the recombinant protein from Escherichia coli . Included are methods to obtain the 15 N-labeled form, which is very useful for detailed structural and dynamic analyses using NMR spectroscopy.
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Physical characterization of a totally synthetic Rubredoxin.
Journal of Inorganic Biochemistry, 1997Co-Authors: Roberto P. Christen, Michael W W Adams, Tatyana Jancic, Zhi H. Zhou, John M. Tomich, Eugene T. SmithAbstract:Abstract The entire polypeptide of hyperthermophilic Pyrococcus furiosus Rubredoxin was synthesized in order to specifically probe structural determinants of protein thermostability. The uv-visible, circular dichroic, electron paramagnetic, and nuclear magnetic resonance spectra, and electrochemical properties, of the native and synthetic proteins were essentially identical. The synthetic protein had a half-life for denaturation of 24 hr at 80°C. The synthetic protein is considerably more thermostable than nonhyperthermophilic Rubredoxins, but not as stable as the native protein. Based on the spectroscopic evidence, it appears that the synthetic protein is incorporating iron properly to form holoprotein, but the peptide still may not be folded correctly.
Toshiko Ichiye - One of the best experts on this subject based on the ideXlab platform.
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understanding Rubredoxin redox sites by density functional theory studies of analogues
Journal of Physical Chemistry A, 2012Co-Authors: Toshiko IchiyeAbstract:Determining the redox energetics of redox site analogues of metalloproteins is essential in unraveling the various contributions to electron transfer properties of these proteins. Since studies of the [4Fe-4S] analogues show that the energies are dependent on the ligand dihedral angles, broken symmetry density functional theory (BS-DFT) with the B3LYP functional and double-ζ basis sets calculations of optimized geometries and electron detachment energies of [1Fe] Rubredoxin analogues are compared to crystal structures and gas-phase photoelectron spectroscopy data, respectively, for [Fe(SCH3)4]0/1-/2-, [Fe(S2-o-xyl)2]0/1-/2-, and Na+[Fe(S2-oxyl) 2]1-/2- in different conformations. In particular, the study of Na+[Fe(S2-o-xyl)2]1-/2- is the only direct comparison of calculated and experimental gas phase detachment energies for the 1-/2- couple found in the Rubredoxins. These results show that variations in the inner sphere energetics by up to ~0.4 eV can be caused by differences in the ligand dihedral angles in either or both redox states. Moreover, these results indicate that the protein stabilizes the conformation that favors reduction. In addition, the free energies and reorganization energies of oxidation and reduction as well as electrostatic potential charges are calculated, which can be used as estimates in continuum electrostatic calculations of electron transfer properties of [1Fe] proteins.
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the molecular determinants of the increased reduction potential of the Rubredoxin domain of rubrerythrin relative to Rubredoxin
Biophysical Journal, 2010Co-Authors: Can E Ergenekan, Justin T Fischer, Toshiko IchiyeAbstract:Based on the crystal structures, three possible sequence determinants have been suggested as the cause of a 285 mV increase in reduction potential of the Rubredoxin domain of rubrerythrin over Rubredoxin by modulating the polar environment around the redox site. Here, electrostatic calculations of crystal structures of Rubredoxin and rubrerythrin and molecular dynamics simulations of Rubredoxin wild-type and mutants are used to elucidate the contributions to the increased reduction potential. Asn160 and His179 in rubrerythrin versus valines in Rubredoxins are predicted to be the major contributors, as the polar side chains contribute significantly to the electrostatic potential in the redox site region. The mutant simulations show both side chains rotating on a nanosecond timescale between two conformations with different electrostatic contributions. Reduction also causes a change in the reduction energy that is consistent with a linear response due to the interesting mechanism of shifting the relative populations of the two conformations. In addition to this, a simulation of a triple mutant indicates the side-chain rotations are approximately anticorrelated so whereas one is in the high potential conformation, the other is in the low potential conformation. However, Ala176 in rubrerythrin versus a leucine in Rubredoxin is not predicted to be a large contributor, because the solvent accessibility increases only slightly in mutant simulations and because it is buried in the interface of the rubrerythrin homodimer.
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leucine 41 is a gate for water entry in the reduction of clostridium pasteurianum Rubredoxin
Protein Science, 2008Co-Authors: Can E Ergenekan, Marly K Eidsness, Toshiko Ichiye, Chulhee KangAbstract:Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced Rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of Rubredoxin should be facilitated. First, expansion of the [Fe—S] cluster and concomitant contraction of the NH • • • S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp Rubredoxin.
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observation of terahertz vibrations in pyrococcus furiosus Rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy interpretation by molecular mechanics
Journal of Inorganic Biochemistry, 2007Co-Authors: Anna Rita Bizzarri, Toshiko Ichiye, Yuming Xiao, Salvatore Cannistraro, Cristian Manzoni, G CerulloAbstract:We have used impulsive coherent vibrational spectroscopy (ICVS) to study the Fe(S-Cys)(4) site in oxidized Rubredoxin (Rd) from Pyrococcus furiosus (Pf). In this experiment, a 15 fs visible laser pulse is used to coherently pump the sample to an excited electronic state, and a second <10 fs pulse is used to probe the change in transmission as a function of the time delay. PfRd was observed to relax to the ground state by a single exponential decay with time constants of approximately 255-275 fs. Superimposed on this relaxation are oscillations caused by coherent excitation of vibrational modes in both excited and ground electronic states. Fourier transformation reveals the frequencies of these modes. The strongest ICV mode with 570 nm excitation is the symmetric Fe-S stretching mode near 310 cm(-1), compared to 313 cm(-1) in the low temperature resonance Raman. If the Rubredoxin is pumped at 520 nm, a set of strong bands occurs between 20 and 110 cm(-1). Finally, there is a mode at approximately 500 cm(-1) which is similar to features near 508 cm(-1) in blue Cu proteins that have been attributed to excited state vibrations. Normal mode analysis using 488 protein atoms and 558 waters gave calculated spectra that are in good agreement with previous nuclear resonance vibrational spectra (NRVS) results. The lowest frequency normal modes are identified as collective motions of the entire protein or large segments of polypeptide. Motion in these modes may affect the polar environment of the redox site and thus tune the electron transfer functions in Rubredoxins.
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The role of backbone stability near Ala44 in the high reduction potential class of Rubredoxins
Proteins, 2006Co-Authors: Chulhee Kang, Toshiko IchiyeAbstract:Rubredoxins may be separated into high and low reduction potential classes, with reduction potentials differing by approximately 50 mV. Our previous work showed that a local shift in the polar backbone due to an A(44) versus V(44) side-chain size causes this reduction potential difference. However, this work also indicated that in the low potential Clostridium pasteurianum (Cp) Rubredoxin, a V(44) --> A(44) mutation causes larger local backbone flexibility, because the V(44) side-chain present in the wild-type (wt) is no longer present to interlock with neighboring residues to stabilize the subsequent G(45). Since Pyrococcus furiosus (Pf) and other high potential Rubredoxins generally have a P(45), it was presumed that a G(45) --> P(45) mutation might stabilize a V(44) --> A(44) mutation in Cp Rubredoxin. Here crystal structure analysis, energy minimization, and molecular dynamics (MD) were performed for wt V(44)G(45), single mutant A(44)G(45) and double mutant A(44)P(45) Cp, and for wt A(44)P(45) Pf Rubredoxins. The local structural, dynamical, and electrostatic properties of Cp gradually approach wt Pf in the order wt Cp to single to double mutant because of greater sequence similarity, as expected. The double mutant A(44)P(45) Cp exhibits increased backbone stability near residue 44 and thus enhances the probability that the backbone dipoles point toward the redox site, which favors an increase in the electrostatic contribution to the reduction potential. It appears that the electrostatic potential of residue 44 and the solvent accessibility to the redox are both determinants for the reduction potentials of homologous Rubredoxins. Overall, these results indicate that an A(44) in a Rubredoxin may require a P(45) for backbone stability whereas a V(44) can accommodate a G(45), since the valine side-chain can interlock with its neighbors.
Jean Legall - One of the best experts on this subject based on the ideXlab platform.
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Structural determinants of protein stabilization by solutes - the importance of the hairpin loop in Rubredoxins
FEBS Journal, 2005Co-Authors: Tiago M. Pais, Jean Legall, Pedro Lamosa, Wagner G. Dos Santos, David L. Turner, Helena SantosAbstract:Despite their high sequence homology, Rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus[Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol66, 1974–1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the Rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas Rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Δ17|29, was determined by 1H-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant Rubredoxins was determined at 90 °C and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas Rubredoxin increased threefold in the presence of 0.1 m diglycerol phosphate, mutant Δ23|29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.
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the genetic organization of desulfovibrio desulphuricans atcc 27774 bacterioferritin and Rubredoxin 2 genes involvement of Rubredoxin in iron metabolism
Molecular Microbiology, 2001Co-Authors: Patricia N Da Costa, Jean Legall, Celia V Romao, Antonio V Xavier, Eurico Melo, Miguel Teixeira, Ligia M SaraivaAbstract:The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having iron-coproporphyrin III as its haem cofactor, as well as a type 2 Rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and Rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and Rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of Rubredoxin-2 tryptophan(s) when mixed with bacterioferritin. In addition, Rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of Rubredoxin-2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that Rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.
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analysis of the desulfovibrio gigas transcriptional unit containing Rubredoxin rd and Rubredoxin oxygen oxidoreductase roo genes and upstream orfs
Biochemical and Biophysical Research Communications, 2001Co-Authors: Gabriela Silva, Jean Legall, Antonio V Xavier, Solange Oliveira, Claudina RodriguespousadaAbstract:Abstract Rubredoxin-oxygen oxidoreductase, an 86-kDa homodimeric flavoprotein, is the final component of a soluble electron transfer chain that couples NADH oxidation with oxygen reduction to water from the sulfate-reducing bacterium Desulfovibrio gigas. A 4.2-kb fragment of D. gigas chromosomal DNA containing the roo gene and the Rubredoxin gene was sequenced. Additional open reading frames designated as ORF-1, ORF-2, and ORF-3 were also identified in this DNA fragment. ORF-1 encodes a protein exhibiting homology to several proteins of the short-chain dehydrogenase/reductase family of enzymes. The N-terminal coenzyme-binding pattern and the active-site pattern characteristic of short chain dehydrogenase/reductase proteins are conserved in ORF-1 product. ORF-2 does not show any significant homology with any known protein, whereas ORF-3 encodes a protein having significant homologies with the branched-chain amino acid transporter AzlC protein family. Northern blot hybridization analysis with rd and roo-specific probes identified a common 1.5-kb transcript, indicating that these two genes are cotranscribed. The transcription start site was identified by primer extension analysis to be a guanidine 87 bp upstream the ATG start codon of Rubredoxin. The transcript size indicates that the rd-roo mRNA terminates downstream the roo-coding unit. Putative −10 and −35 regulator regions of a ς70-type promoter, having similarity with E. coli ς70 promoter elements, are found upstream the transcription start site. Rubredoxin-oxygen oxidoreductase and Rubredoxin genes are shown to be constitutively and abundantly expressed. Using the data available from different prokaryotic genomes, the Rubredoxin genomic organization and the first tentative to understand the phylogenetic relationships among the flavoprotein family are reported in this study.
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Characterisation of a new Rubredoxin isolated from Desulfovibrio desulfuricans 27774: definition of a new family of Rubredoxins
FEBS Letters, 1998Co-Authors: Jean Legall, Antonio V Xavier, Cláudio M. Gomes, Vera Braga, Isabel Pacheco, Manuela Regalla, Miguel TeixeiraAbstract:Abstract A new Rubredoxin from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as terminal electron acceptor, was isolated and characterised. The protein is an 8.5 kDa monomer containing one iron atom per molecule, with a reduction potential of 25±5 mV at pH 7.6. Like the recombinant Rdl protein from D. vulgaris, expressed in Escherichia coli [Lumpio, H.L., Shenvi, N.V., Garg, R.P., Summers, A.O. and Kurtz, D.M., J. Bacteriol. 179 (1997) 4607–4615], it contains an unusual spacing of four amino acids between the first two of the iron coordinating cysteinyl residues. This difference is reflected in the structure of the iron centre, as observed by visible and EPR spectroscopies. All together, these features make these proteins the first members of a new family of Rubredoxins.
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Rubredoxin in crystalline state
Methods in Enzymology, 1994Co-Authors: L C Sieker, R E Stenkamp, Jean LegallAbstract:Publisher Summary This chapter focuses on Rubredoxin (Rd) in crystalline state, which is regarded as one of the simplest iron proteins. Rds are composed of 45 to 54 amino-acid residues with molecular weights ranging from 5000 to 6000 and contain one iron atom liganded by four cysteine residues. The iron center can be reversibly reduced at a redox potential near 0 mV. Rds are divided into three categories: (1) Rds from sulfate-reducing Desulfovibrio species, (2) Rds from a mixed assortment of bacteria, and (3) thermophilic Rds. The redox potentials of Rds isolated from sulfate-reducing bacteria (SRB) are relatively high. The amino-acid sequences of all Rubredoxins show two sets of the -C- x - y -C-G- z - sequence around the iron center, where each cysteine is a ligand to the iron atom. Lys-46 is the only invariant hydrophilic residue in all the Rds. Except for the Desulfovibrio vulgaris ( D. vulgaris ) Rd structure, the crystal structures of the other Rds show that Lys-46 extends across to the neighboring chain, making an H bond to the carbonyl oxygens of residue 30 and residue 33, presumably contributing to the stability of the molecule.
Donald M. Kurtz - One of the best experts on this subject based on the ideXlab platform.
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thermal stability of the fe scys 4 site in clostridium pasteurianum Rubredoxin contributions of the local environment and cys ligand protonation
Journal of Biological Inorganic Chemistry, 2002Co-Authors: Francesco Bonomi, Donald M. Kurtz, Amy E Burden, Marly K Eidsness, Dimitrios Fessas, S Iametti, Stefania Mazzini, Robert A Scott, Qiandong ZengAbstract:Thermal denaturation of the mesophilic Rubredoxin from Clostridium pasteurianum occurs through a number of temperature-dependent steps, the last and irreversible one being release of iron from the [Fe2+(SCys)4] site. We show here that thermally induced [Fe2+(SCys)4] site destruction is largely determined by the local environment, and not directly connected to thermostability of the native polypeptide fold of Rubredoxin. Hydrophobic residues on the protein surface, V8 and L41, that shield the [Fe(SCys)4] site from solvent and form N-H...S hydrogen bonds to the metal-coordinating sulfurs, were mutated to residues with both uncharged and charged side chains. On these mutated Rubredoxins the temperature dependence was measured for: (1) global unfolding of the protein by NMR, (2) loss of Fe2+ at various ionic strengths and pH values, (3) the rates of non-denaturing displacement of Fe2+ by Cd2+ or Zn2+. For reversible temperature-dependent changes in the global protein folding that occur prior to loss of iron, no thermostability differences were found among the wild-type, V8A, V8D, L41R, and L41D Rubredoxins. However, for irreversible loss of iron from the [Fe2+(SCys)4] site, relative to the wild-type protein, L41R was more thermostable, V8A was somewhat less thermostable, and the acidic mutants L41D, V8D and [V8D, L41D] showed dramatically lowered thermostability. Lower pH facilitated – both kinetically and thermodynamically – thermally induced iron release, likely through protonation of ligand cysteines' thiols. For all of the Rubredoxins a direct correlation was found between the midpoint temperature for thermally induced Fe2+ loss and the rate of non-denaturing Fe2+ displacement by Cd2+ or Zn2+ at room temperature. A mechanism is proposed involving transient movement of residue-8 and -41 side chains, allowing, and, in the case of negatively charged side chains, also facilitating, attack of a ligand cysteine by the incoming positively charged species (H+, Cd2+, or Zn2+). Thus, localized charge density and solvent accessibility modulate the stability of Fe2+ ligation in Rubredoxin. However, the reduced [Fe(SCys)4] site does not control the thermostability of the native polypeptide fold of Rubredoxin.
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a role for Rubredoxin in oxidative stress protection in desulfovibrio vulgaris catalytic electron transfer to rubrerythrin and two iron superoxide reductase
Archives of Biochemistry and Biophysics, 2001Co-Authors: Eric D Coulter, Donald M. KurtzAbstract:Abstract Desulfovibrio vulgaris Rubredoxin, which contains a single [Fe(SCys) 4 ] site, is shown to be a catalytically competent electron donor to two enzymes from the same organism, namely, rubrerythrin and two-iron superoxide reductase (a.k.a. Rubredoxin oxidoreductase or desulfoferrodoxin). These two enzymes have been implicated in catalytic reduction of hydrogen peroxide and superoxide, respectively, during periods of oxidative stress in D. vulgaris, but their proximal electron donors had not been characterized. We further demonstrate the incorrectness of a previous report that Rubredoxin is not an electron donor to the superoxide reductase and describe convenient assays for demonstrating the catalytic competence of all three proteins in their respective functions. Rubrerythrin is shown to be an efficient Rubredoxin peroxidase in which the rubedoxin:hydrogen peroxide redox stoichiometry is 2:1 mol:mol. Using spinach ferredoxin-NADP + oxidoreductase (FNR) as an artificial, but proficient, NADPH:Rubredoxin reductase, Rubredoxin was further found to catalyze rapid and complete reduction of all Fe 3+ to Fe 2+ in rubrerythrin by NADPH under anaerobic conditions. The combined system, FNR/Rubredoxin/rubrerythrin, was shown to function as a catalytically competent NADPH peroxidase. Another small Rubredoxin-like D. vulgaris protein, Rdl, could not substitute for Rubredoxin as a peroxidase substrate of rubrerythrin. Similarly, D. vulgaris Rubredoxin was demonstrated to efficiently catalyze reduction of D. vulgaris two-iron superoxide reductase and, when combined with FNR, to function as an NADPH:superoxide oxidoreductase. We suggest that, during periods of oxidative stress, Rubredoxin could divert electron flow from the electron transport chain of D. vulgaris to rubrerythrin and superoxide reductase, thereby simultaneously protecting autoxidizable redox enzymes and lowering intracellular hydrogen peroxide and superoxide levels.
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five gene cluster in clostridium thermoaceticum consisting of two divergent operons encoding Rubredoxin oxidoreductase Rubredoxin and rubrerythrin type a flavoprotein high molecular weight Rubredoxin
Journal of Bacteriology, 2001Co-Authors: Eric D Coulter, Donald M. Kurtz, Lars G LjungdahlAbstract:A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (Rubredoxin), rbo (Rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight Rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, Rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.
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Direct metal ion substitution at the [M(SCys)4]2- site of Rubredoxin
Journal of Biological Inorganic Chemistry, 1998Co-Authors: Franco Bonomi, Stefania Iametti, Donald M. Kurtz, Enzio Ragg, Kimberly A. RichieAbstract:The single Fe(II) in reduced Rubredoxin from Clostridium pasteurianum was found to be quantitatively displaced by either Cd2+ or Zn2+ when a modest molar excess of the substituting metal salt was anaerobically incubated with the reduced Rubredoxin under mild conditions, namely, room temperature, pH 5.4–8.4, and no protein denaturants. Under the same conditions, cadmium-for-zinc substitution was also achieved upon aerobic incubation of the zinc-substituted Rubredoxin with a modest molar excess of Cd2+. Displacements of Fe(II) from the reduced Rubredoxin were not observed upon anaerobic incubation with Ni2+, Co2+, or VO2+ salts, and no reaction with any of the divalent metal ions was observed for the oxidized [Fe(III)] Rubredoxin. Fe(II) could not be re-inserted into the Zn- or Cd-substituted Rubredoxins without resorting to protein denaturation. 1H and 113Cd NMR experiments showed that the cadmium-substituted Rubredoxin prepared by the non-denaturing substitution method retained the pseudotetrahedral M(SCys)4 coordination geometry and secondary structural elements characteristic of the native Rubredoxin, and that "unzipping" of the β-sheet did not occur during metal substitution. Rates of Fe(II) displacement by M2+ (M=Cd or Zn) increased with increasing M2+/Rubredoxin ratio, decreasing pH, and lower ionic strength. The substitution rates were faster for M=Cd than for M=Zn. Rates of Cd2+ substitution into a V8A-mutated Rubredoxin were significantly faster than for the wild-type protein. The side-chain of V8 is on the protein surface and close to the metal-ligating Cys42Sγ at the M(SCys)4 site. Therefore, the rate-limiting step in the substitution process is suggested to involve direct attack of the [M(SCys)4]2– site by the incoming M2+, without global unfolding of the protein. Implications of these results for metal ion incorporation into Rubredoxins in vivo are discussed.
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Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum Rubredoxin
Protein Engineering, 1992Co-Authors: Marly K Eidsness, Donald M. Kurtz, Sonia E. O'dell, Robert L. Robson, Robert A ScottAbstract:A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum Rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E. coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) Rubredoxin and zinc-substituted Rubredoxin, with the latter amounting to approximately 70% of the total Rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holoRubredoxin are characteristic of the native Rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-... (63%), blocked (30%) and Met-Lys-... (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-... and nMet-Lys-..., where nMet represents an amino-blocked methionine residue.
Antonio V Xavier - One of the best experts on this subject based on the ideXlab platform.
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the genetic organization of desulfovibrio desulphuricans atcc 27774 bacterioferritin and Rubredoxin 2 genes involvement of Rubredoxin in iron metabolism
Molecular Microbiology, 2001Co-Authors: Patricia N Da Costa, Jean Legall, Celia V Romao, Antonio V Xavier, Eurico Melo, Miguel Teixeira, Ligia M SaraivaAbstract:The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having iron-coproporphyrin III as its haem cofactor, as well as a type 2 Rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and Rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and Rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of Rubredoxin-2 tryptophan(s) when mixed with bacterioferritin. In addition, Rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of Rubredoxin-2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that Rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.
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analysis of the desulfovibrio gigas transcriptional unit containing Rubredoxin rd and Rubredoxin oxygen oxidoreductase roo genes and upstream orfs
Biochemical and Biophysical Research Communications, 2001Co-Authors: Gabriela Silva, Jean Legall, Antonio V Xavier, Solange Oliveira, Claudina RodriguespousadaAbstract:Abstract Rubredoxin-oxygen oxidoreductase, an 86-kDa homodimeric flavoprotein, is the final component of a soluble electron transfer chain that couples NADH oxidation with oxygen reduction to water from the sulfate-reducing bacterium Desulfovibrio gigas. A 4.2-kb fragment of D. gigas chromosomal DNA containing the roo gene and the Rubredoxin gene was sequenced. Additional open reading frames designated as ORF-1, ORF-2, and ORF-3 were also identified in this DNA fragment. ORF-1 encodes a protein exhibiting homology to several proteins of the short-chain dehydrogenase/reductase family of enzymes. The N-terminal coenzyme-binding pattern and the active-site pattern characteristic of short chain dehydrogenase/reductase proteins are conserved in ORF-1 product. ORF-2 does not show any significant homology with any known protein, whereas ORF-3 encodes a protein having significant homologies with the branched-chain amino acid transporter AzlC protein family. Northern blot hybridization analysis with rd and roo-specific probes identified a common 1.5-kb transcript, indicating that these two genes are cotranscribed. The transcription start site was identified by primer extension analysis to be a guanidine 87 bp upstream the ATG start codon of Rubredoxin. The transcript size indicates that the rd-roo mRNA terminates downstream the roo-coding unit. Putative −10 and −35 regulator regions of a ς70-type promoter, having similarity with E. coli ς70 promoter elements, are found upstream the transcription start site. Rubredoxin-oxygen oxidoreductase and Rubredoxin genes are shown to be constitutively and abundantly expressed. Using the data available from different prokaryotic genomes, the Rubredoxin genomic organization and the first tentative to understand the phylogenetic relationships among the flavoprotein family are reported in this study.
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Characterisation of a new Rubredoxin isolated from Desulfovibrio desulfuricans 27774: definition of a new family of Rubredoxins
FEBS Letters, 1998Co-Authors: Jean Legall, Antonio V Xavier, Cláudio M. Gomes, Vera Braga, Isabel Pacheco, Manuela Regalla, Miguel TeixeiraAbstract:Abstract A new Rubredoxin from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as terminal electron acceptor, was isolated and characterised. The protein is an 8.5 kDa monomer containing one iron atom per molecule, with a reduction potential of 25±5 mV at pH 7.6. Like the recombinant Rdl protein from D. vulgaris, expressed in Escherichia coli [Lumpio, H.L., Shenvi, N.V., Garg, R.P., Summers, A.O. and Kurtz, D.M., J. Bacteriol. 179 (1997) 4607–4615], it contains an unusual spacing of four amino acids between the first two of the iron coordinating cysteinyl residues. This difference is reflected in the structure of the iron centre, as observed by visible and EPR spectroscopies. All together, these features make these proteins the first members of a new family of Rubredoxins.
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studies on the redox centers of the terminal oxidase from desulfovibrio gigas and evidence for its interaction with Rubredoxin
Journal of Biological Chemistry, 1997Co-Authors: Cláudio M. Gomes, Antonio V Xavier, Gabriela Silva, Solange Oliveira, Claudina Rodriguespousada, J Legall, Miguel TeixeiraAbstract:Abstract Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducerDesulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at ∼2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 ± 15 mV for the quinone/semiquinone couple and at −130 ± 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is −350 ± 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from Rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that Rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a Rubredoxin in a sulfate-reducing bacterium is presented.
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purification and characterization of an nadh Rubredoxin oxidoreductase involved in the utilization of oxygen by desulfovibrio gigas
FEBS Journal, 1993Co-Authors: Liang Chen, Jean Legall, Paula Fareleira, Helena Santos, Antonio V XavierAbstract:An NADH–Rubredoxin oxidoreductase previously isolated from Desulfovibrio gigas [LeGall, J. (1968) Ann. Inst. Pasteur 114, 109–115] has now been fully purified and further characterized. It contains two subunits of 27 kDa and 32 kDa. With two mid-point redox potentials of –295 mV and –325mV, this FMN- and FAD-containing protein can induce the specific reduction of D. gigas Rubredoxin. In contrast, Rubredoxins from the other Desulfovibrio species or desulforedoxin from D. gigas show very low reaction rates with the same enzyme. The phylogenetic significance of the narrow specificity of the enzyme toward the Rubredoxin from the same organism is discussed. The purified enzyme has NADH oxidase activity with H2O2 as a final product of O2 reduction. The reaction is half-inhibited by 4.2μM p-chloromercuribenzoate, whereas cyanide and azide are not significant inhibitors in this reaction. The role of this protein as a part of the enzymic equipment that allows the formation of ATP in the presence of oxygen from the degradation of carbon reserves is discussed.
