31 results on '"Jenney, Francis E."'
Search Results
2. Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.
- Author
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Chandrayan SK, McTernan PM, Hopkins RC, Sun J, Jenney FE Jr, and Adams MW
- Subjects
- Archaeal Proteins genetics, Biofuels, Catalytic Domain physiology, Cytoplasm genetics, Gene Expression, Hydrogenase genetics, Maltose metabolism, Maltose pharmacology, Operon physiology, Pyrococcus furiosus genetics, Pyrococcus furiosus growth & development, Sweetening Agents metabolism, Sweetening Agents pharmacology, Archaeal Proteins biosynthesis, Cytoplasm enzymology, Hydrogen metabolism, Hydrogenase biosynthesis, Metabolic Engineering, Pyrococcus furiosus enzymology
- Abstract
The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.
- Published
- 2012
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3. Dynamics of the [4Fe-4S] cluster in Pyrococcus furiosus D14C ferredoxin via nuclear resonance vibrational and resonance Raman spectroscopies, force field simulations, and density functional theory calculations.
- Author
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Mitra D, Pelmenschikov V, Guo Y, Case DA, Wang H, Dong W, Tan ML, Ichiye T, Jenney FE, Adams MW, Yoda Y, Zhao J, and Cramer SP
- Subjects
- Bacterial Proteins metabolism, Ferredoxins metabolism, Iron metabolism, Magnetic Resonance Spectroscopy, Models, Molecular, Oxidation-Reduction, Spectrum Analysis, Raman, Sulfur metabolism, Bacterial Proteins chemistry, Ferredoxins chemistry, Iron chemistry, Pyrococcus furiosus metabolism, Sulfur chemistry
- Abstract
We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ∼400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.
- Published
- 2011
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4. Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases.
- Author
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Lipscomb GL, Stirrett K, Schut GJ, Yang F, Jenney FE Jr, Scott RA, Adams MW, and Westpheling J
- Subjects
- Archaeal Proteins genetics, Hydrogenase genetics, Pyrococcus furiosus enzymology, Gene Deletion, Genetics, Microbial methods, Pyrococcus furiosus genetics, Recombination, Genetic, Transformation, Genetic
- Abstract
In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.
- Published
- 2011
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5. Homologous expression of a subcomplex of Pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase.
- Author
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Hopkins RC, Sun J, Jenney FE Jr, Chandrayan SK, McTernan PM, and Adams MW
- Subjects
- Catalysis, Catalytic Domain, Protein Binding, Recombination, Genetic, Hydrogenase metabolism, Pyrococcus furiosus enzymology, Pyruvate Synthase metabolism
- Abstract
Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.
- Published
- 2011
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6. Microbial metalloproteomes are largely uncharacterized.
- Author
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Cvetkovic A, Menon AL, Thorgersen MP, Scott JW, Poole FL 2nd, Jenney FE Jr, Lancaster WA, Praissman JL, Shanmukh S, Vaccaro BJ, Trauger SA, Kalisiak E, Apon JV, Siuzdak G, Yannone SM, Tainer JA, and Adams MW
- Subjects
- Bacterial Proteins chemistry, Chromatography, Liquid, Escherichia coli chemistry, Metals chemistry, Metals metabolism, Proteome chemistry, Proteomics, Pyrococcus furiosus metabolism, Sulfolobus solfataricus chemistry, Tandem Mass Spectrometry, Bacterial Proteins analysis, Metalloproteins analysis, Metalloproteins chemistry, Metals analysis, Proteome analysis, Pyrococcus furiosus chemistry
- Abstract
Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.
- Published
- 2010
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7. Oxidative stress protection and the repair response to hydrogen peroxide in the hyperthermophilic archaeon Pyrococcus furiosus and in related species.
- Author
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Strand KR, Sun C, Li T, Jenney FE Jr, Schut GJ, and Adams MW
- Subjects
- Archaeal Proteins genetics, DNA, Archaeal genetics, Gene Expression Regulation, Archaeal, Hemerythrin genetics, Hemerythrin metabolism, Hydrogen Peroxide pharmacology, NADP metabolism, Open Reading Frames, Oxidation-Reduction, Oxidoreductases genetics, Oxidoreductases metabolism, Peroxiredoxins genetics, Peroxiredoxins metabolism, Pyrococcus furiosus enzymology, Pyrococcus furiosus genetics, Pyrococcus furiosus growth & development, Rubredoxins genetics, Rubredoxins metabolism, Thermococcus genetics, Thermococcus metabolism, Transcription, Genetic, Up-Regulation, Archaeal Proteins metabolism, DNA Repair, Hydrogen Peroxide metabolism, Oxidative Stress genetics, Pyrococcus furiosus metabolism
- Abstract
Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100 degrees C. Addition of H(2)O(2) (0.5 mM) to a growing culture resulted in the cessation of growth with a 2-h lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function, while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H(2)O(2) were not more resistant to the subsequent addition of H(2)O(2) (0.5-5.0 mM). Consequently, there is little if any induced protective response to peroxide. The organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin, and alkyl hydroperoxide reductase. Related hyperthermophiles contain homologs of the proteins involved in the constitutive protective mechanism but these organisms were more sensitive to peroxide than P. furiosus and lack several of its peroxide-responsive ORFs.
- Published
- 2010
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8. Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.
- Author
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Sun J, Hopkins RC, Jenney FE, McTernan PM, and Adams MW
- Subjects
- Amino Acid Sequence, Base Sequence, Escherichia coli genetics, Molecular Sequence Data, Plasmids chemistry, Plasmids genetics, Promoter Regions, Genetic genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, beta-Galactosidase genetics, Biofuels microbiology, Escherichia coli metabolism, Hydrogenase metabolism, NADP metabolism, Pyrococcus furiosus enzymology
- Abstract
Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.
- Published
- 2010
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9. Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome.
- Author
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Menon AL, Poole FL 2nd, Cvetkovic A, Trauger SA, Kalisiak E, Scott JW, Shanmukh S, Praissman J, Jenney FE Jr, Wikoff WR, Apon JV, Siuzdak G, and Adams MW
- Subjects
- Amino Acids metabolism, Archaeal Proteins isolation & purification, Cytoplasm metabolism, Protein Denaturation, Protein Multimerization, Archaeal Proteins analysis, Chemical Fractionation methods, Multiprotein Complexes analysis, Proteome analysis, Pyrococcus furiosus metabolism
- Abstract
Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.
- Published
- 2009
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10. The first agmatine/cadaverine aminopropyl transferase: biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus.
- Author
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Cacciapuoti G, Porcelli M, Moretti MA, Sorrentino F, Concilio L, Zappia V, Liu ZJ, Tempel W, Schubot F, Rose JP, Wang BC, Brereton PS, Jenney FE, and Adams MW
- Subjects
- Agmatine metabolism, Alkyl and Aryl Transferases biosynthesis, Archaea, Archaeal Proteins biosynthesis, Cadaverine metabolism, Genes, Archaeal, Hot Temperature, Pyrococcus furiosus genetics, Spermidine Synthase chemistry, Spermidine Synthase metabolism, Polyamines metabolism, Pyrococcus furiosus enzymology, Pyrococcus furiosus physiology
- Abstract
We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.
- Published
- 2007
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11. Structure of the hypothetical protein PF0899 from Pyrococcus furiosus at 1.85 A resolution.
- Author
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Kelley LL, Dillard BD, Tempel W, Chen L, Shaw N, Lee D, Newton MG, Sugar FJ, Jenney FE Jr, Lee HS, Shah C, Poole FL 3rd, Adams MW, Richardson JS, Richardson DC, Liu ZJ, Wang BC, and Rose J
- Subjects
- Archaeal Proteins genetics, Archaeal Proteins isolation & purification, Cloning, Molecular, Crystallization, Crystallography, X-Ray, Open Reading Frames genetics, Protein Structure, Secondary, Pyrococcus furiosus genetics, Archaeal Proteins chemistry, Pyrococcus furiosus chemistry
- Abstract
The hypothetical protein PF0899 is a 95-residue peptide from the hyperthermophilic archaeon Pyrococcus furiosus that represents a gene family with six members. P. furiosus ORF PF0899 has been cloned, expressed and crystallized and its structure has been determined by the Southeast Collaboratory for Structural Genomics (http://www.secsg.org). The structure was solved using the SCA2Structure pipeline from multiple data sets and has been refined to 1.85 A against the highest resolution data set collected (a presumed gold derivative), with a crystallographic R factor of 21.0% and R(free) of 24.0%. The refined structure shows some structural similarity to a wedge-shaped domain observed in the structure of the major capsid protein from bacteriophage HK97, suggesting that PF0899 may be a structural protein.
- Published
- 2007
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12. Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy--interpretation by molecular mechanics.
- Author
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Tan ML, Bizzarri AR, Xiao Y, Cannistraro S, Ichiye T, Manzoni C, Cerullo G, Adams MW, Jenney FE Jr, and Cramer SP
- Subjects
- Fourier Analysis, Models, Chemical, Protein Structure, Secondary radiation effects, Spectrum Analysis, Raman, Structure-Activity Relationship, Vibration, Iron chemistry, Magnetic Resonance Spectroscopy methods, Pyrococcus furiosus chemistry, Rubredoxins chemistry, Spectrophotometry, Infrared methods
- Abstract
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.
- Published
- 2007
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13. Structural and transcriptional analyses of a purine nucleotide-binding protein from Pyrococcus furiosus: a component of a novel, membrane-bound multiprotein complex unique to this hyperthermophilic archaeon.
- Author
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Gerwe B, Kelley LL, Dillard BD, Lai T, Liu ZJ, Tempel W, Chen L, Habel J, Lee D, Jenney FE Jr, Sugar FJ, Richardson JS, Richardson DC, Newton MG, Wang BC, Adams MW, and Rose JP
- Subjects
- Amino Acid Sequence, Biological Transport, Crystallography, X-Ray, Genome, Archaeal, Genomics, Models, Biological, Molecular Conformation, Molecular Sequence Data, Nucleotide Transport Proteins physiology, Protein Conformation, Proteins metabolism, Sequence Homology, Amino Acid, Transcription, Genetic, Gene Expression Regulation, Archaeal, Multiprotein Complexes chemistry, Nucleotide Transport Proteins chemistry, Proteins chemistry, Pyrococcus furiosus metabolism
- Abstract
The open-reading frame PF0895 in the genome of the hyperthermophilic archaeon, Pyrococcus furiosus, encodes a 206-residue protein (M(R )23,152). The structure of the recombinant protein was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. It has been refined to 1.70 A with a crystallographic R and R(free )values of 19.7% and 22.3%, respectively. The PF0895 structure is similar to those of the ATP binding cassettes observed in the ABC transporter family. However, bioinformatics and molecular analyses indicate that PF0895 is not part of the expected five-gene operon that encodes a typical prokaryotic solute-binding ABC transporter. Rather, transcriptional profiling data show that PF0895 is part of a novel four-gene operon (PF0895-PF0896-PF0897-PF0897.1) where only PF0895 has homologs in other organisms. Interestingly, from genome analysis, P. furiosus itself contains a second version of this complex, encoded by PF1090-PF1093. From the structural studies we can only conclude that one of the subunits of this novel membrane complex, PF0895, and its homolog PF1090, likely bind a purine nucleotide. PF0895 is therefore predicted to be part of a membrane-bound multiprotein complex unrelated to ABC transporters that is so far unique to P. furiosus. It appears to play a role in the stress response, as its expression is down regulated when the organism is subjected to cold-shock, where cells are transferred from 95 degrees C, near the optimal growth temperature, to 72 degrees C, near the minimal growth temperature. The related PF1090-containing operon is unaffected by cold-shock and is independently regulated.
- Published
- 2007
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14. Transcriptional and biochemical analysis of starch metabolism in the hyperthermophilic archaeon Pyrococcus furiosus.
- Author
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Lee HS, Shockley KR, Schut GJ, Conners SB, Montero CI, Johnson MR, Chou CJ, Bridger SL, Wigner N, Brehm SD, Jenney FE Jr, Comfort DA, Kelly RM, and Adams MW
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- Archaeal Proteins genetics, Bacterial Proteins genetics, DNA, Archaeal chemistry, DNA, Archaeal genetics, Glucose metabolism, Glucosephosphates metabolism, Glucosyltransferases genetics, Glycogen Debranching Enzyme System genetics, Glycoside Hydrolases genetics, Monosaccharide Transport Proteins genetics, Oligonucleotide Array Sequence Analysis, Pyrococcus furiosus genetics, Sequence Analysis, DNA, Substrate Specificity, Gene Expression Profiling, Gene Expression Regulation, Archaeal, Pyrococcus furiosus metabolism, Starch metabolism
- Abstract
Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these alpha-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-alpha-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of alpha-glucan substrates by P. furiosus.
- Published
- 2006
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15. Defining genes in the genome of the hyperthermophilic archaeon Pyrococcus furiosus: implications for all microbial genomes.
- Author
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Poole FL 2nd, Gerwe BA, Hopkins RC, Schut GJ, Weinberg MV, Jenney FE Jr, and Adams MW
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- Archaeal Proteins chemistry, Archaeal Proteins genetics, Computational Biology, Escherichia coli genetics, Gene Expression Profiling, Genomics methods, Protein Biosynthesis, Pyrococcus furiosus physiology, RNA, Archaeal analysis, Recombinant Proteins biosynthesis, Transcription, Genetic, Databases, Nucleic Acid standards, Genes, Archaeal, Genome, Archaeal, Open Reading Frames, Pyrococcus furiosus genetics
- Abstract
The original genome annotation of the hyperthermophilic archaeon Pyrococcus furiosus contained 2,065 open reading frames (ORFs). The genome was subsequently automatically annotated in two public databases by the Institute for Genomic Research (TIGR) and the National Center for Biotechnology Information (NCBI). Remarkably, more than 500 of the originally annotated ORFs differ in size in the two databases, many very significantly. For example, more than 170 of the predicted proteins differ at their N termini by more than 25 amino acids. Similar discrepancies were observed in the TIGR and NCBI databases with the other archaeal and bacterial genomes examined. In addition, the two databases contain 60 (NCBI) and 221 (TIGR) ORFs not present in the original annotation of P. furiosus. In the present study we have experimentally assessed the validity of 88 previously unannotated ORFs. Transcriptional analyses showed that 11 of 61 ORFs examined were expressed in P. furiosus when grown at either 95 or 72 degrees C. In addition, 7 of 54 ORFs examined yielded heat-stable recombinant proteins when they were expressed in Escherichia coli, although only one of the seven ORFs was expressed in P. furiosus under the growth conditions tested. It is concluded that the P. furiosus genome contains at least 17 ORFs not previously recognized in the original annotation. This study serves to highlight the discrepancies in the public databases and the problems of accurately defining the number and sizes of ORFs within any microbial genome.
- Published
- 2005
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16. Normal mode analysis of Pyrococcus furiosus rubredoxin via nuclear resonance vibrational spectroscopy (NRVS) and resonance raman spectroscopy.
- Author
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Xiao Y, Wang H, George SJ, Smith MC, Adams MW, Jenney FE Jr, Sturhahn W, Alp EE, Zhao J, Yoda Y, Dey A, Solomon EI, and Cramer SP
- Subjects
- Iron Isotopes, Oxidation-Reduction, Protein Conformation, Protein Structure, Secondary, Vibration, Magnetic Resonance Spectroscopy methods, Models, Chemical, Pyrococcus furiosus chemistry, Rubredoxins chemistry, Spectrum Analysis, Raman methods
- Abstract
We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(S(cys))(4) site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). The oxidized form has also been investigated by resonance Raman spectroscopy. In the oxidized Rd NRVS, strong asymmetric Fe-S stretching modes are observed between 355 and 375 cm(-1); upon reduction these modes shift to 300-320 cm(-1). This is the first observation of Fe-S stretching modes in a reduced Rd. The peak in S-Fe-S bend mode intensity is at approximately 150 cm(-1) for the oxidized protein and only slightly lower in the reduced case. A third band occurs near 70 cm(-1) for both samples; this is assigned primarily as a collective motion of entire cysteine residues with respect to the central Fe. The (57)Fe partial vibrational density of states (PVDOS) were interpreted by normal mode analysis with optimization of Urey-Bradley force fields. The three main bands were qualitatively reproduced using a D(2)(d) Fe(SC)(4) model. A C(1) Fe(SCC)(4) model based on crystallographic coordinates was then used to simulate the splitting of the asymmetric stretching band into at least 3 components. Finally, a model employing complete cysteines and 2 additional neighboring atoms was used to reproduce the detailed structure of the PVDOS in the Fe-S stretch region. These results confirm the delocalization of the dynamic properties of the redox-active Fe site. Depending on the molecular model employed, the force constant K(Fe-S) for Fe-S stretching modes ranged from 1.24 to 1.32 mdyn/A. K(Fe-S) is clearly diminished in reduced Rd; values from approximately 0.89 to 1.00 mdyn/A were derived from different models. In contrast, in the final models the force constants for S-Fe-S bending motion, H(S-Fe-S), were 0.18 mdyn/A for oxidized Rd and 0.15 mdyn/A for reduced Rd. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.
- Published
- 2005
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17. In vitro reconstitution of an NADPH-dependent superoxide reduction pathway from Pyrococcus furiosus.
- Author
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Grunden AM, Jenney FE Jr, Ma K, Ji M, Weinberg MV, and Adams MW
- Subjects
- Amino Acid Sequence, Base Sequence, Biophysical Phenomena, Biophysics, DNA, Archaeal genetics, Genes, Archaeal, Hydrogen Peroxide metabolism, Kinetics, Molecular Sequence Data, NADH, NADPH Oxidoreductases chemistry, NADH, NADPH Oxidoreductases genetics, Oxidation-Reduction, Pyrococcus furiosus genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, NADH, NADPH Oxidoreductases metabolism, NADP metabolism, Pyrococcus furiosus metabolism, Superoxides metabolism
- Abstract
A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of -173 mV at 23 degrees C (-193 mV at 80 degrees C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80 degrees C) and low (23 degrees C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.
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- 2005
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18. Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.
- Author
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Story SV, Shah C, Jenney FE Jr, and Adams MW
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- Amino Acid Sequence, Aminopeptidases isolation & purification, Archaeal Proteins genetics, Archaeal Proteins isolation & purification, Archaeal Proteins metabolism, Catalysis, Molecular Sequence Data, Recombinant Proteins genetics, Recombinant Proteins metabolism, Zinc metabolism, Aminopeptidases genetics, Aminopeptidases metabolism, Lysine metabolism, Pyrococcus furiosus enzymology, Pyrococcus furiosus genetics
- Abstract
Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of L-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 +/- 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100 degrees C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100 degrees C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.
- Published
- 2005
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19. Protein production and crystallization at SECSG -- an overview.
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Wang BC, Adams MW, Dailey H, DeLucas L, Luo M, Rose J, Bunzel R, Dailey T, Habel J, Horanyi P, Jenney FE Jr, Kataeva I, Lee HS, Li S, Li T, Lin D, Liu ZJ, Luan CH, Mayer M, Nagy L, Newton MG, Ng J, Poole FL 2nd, Shah A, Shah C, Sugar FJ, and Xu H
- Subjects
- Animals, Humans, Proteins metabolism, Proteomics instrumentation, Proteomics organization & administration, Caenorhabditis elegans chemistry, Crystallization methods, Databases, Protein, Proteins chemistry, Proteins isolation & purification, Proteomics methods, Pyrococcus furiosus chemistry
- Abstract
Using a high degree of automation, the Southeast Collaboratory for Structural Genomics (SECSG) has developed high throughput pipelines for protein production, and crystallization using a two-tiered approach. Primary, or tier-1, protein production focuses on producing proteins for members of large Pfam families that lack a representative structure in the Protein Data Bank. Target genomes are Pyrococcus furiosus and Caenorhabditis elegans. Selected human proteins are also under study. Tier-2 protein production, or target rescue, focuses on those tier-1 proteins, which either fail to crystallize or give poorly diffracting crystals. This two tier approach is more efficient since it allows the primary protein production groups to focus on the production of new targets while the tier-2 efforts focus on providing additional sample for further studies and modified protein for structure determination. Both efforts feed the SECSG high throughput crystallization pipeline, which is capable of screening over 40 proteins per week. Details of the various pipelines in use by the SECSG for protein production and crystallization, as well as some examples of target rescue are described.
- Published
- 2005
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20. Comparison of small- and large-scale expression of selected Pyrococcus furiosus genes as an aid to high-throughput protein production.
- Author
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Sugar FJ, Jenney FE Jr, Poole FL 2nd, Brereton PS, Izumi M, Shah C, and Adams MW
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- Bacterial Proteins metabolism, Culture Media, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Open Reading Frames genetics, Recombinant Proteins metabolism, Bacterial Proteins isolation & purification, Gene Expression, Proteomics methods, Pyrococcus furiosus genetics, Recombinant Proteins isolation & purification
- Abstract
As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.
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- 2005
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21. Rubrerythrin from the hyperthermophilic archaeon Pyrococcus furiosus is a rubredoxin-dependent, iron-containing peroxidase.
- Author
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Weinberg MV, Jenney FE Jr, Cui X, and Adams MW
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins isolation & purification, Catalysis, Ferredoxins chemistry, Ferredoxins isolation & purification, Hemerythrin, NAD metabolism, Recombinant Proteins isolation & purification, Bacterial Proteins metabolism, Ferredoxins metabolism, Peroxidases metabolism, Pyrococcus furiosus enzymology, Rubredoxins metabolism
- Abstract
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 degrees C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 micromol of rubredoxin oxidized/min/mg with apparent K(m) values of 35 and 70 microM 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 micromol of H(2)O(2) reduced/min/mg of rubrerythrin at 85 degrees 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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- 2004
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22. Structural genomics of Pyrococcus furiosus: X-ray crystallography reveals 3D domain swapping in rubrerythrin.
- Author
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Tempel W, Liu ZJ, Schubot FD, Shah A, Weinberg MV, Jenney FE Jr, Arendall WB 3rd, Adams MW, Richardson JS, Richardson DC, Rose JP, and Wang BC
- Subjects
- Amino Acid Sequence, Binding Sites, Crystallography, X-Ray, Hemerythrin, Molecular Sequence Data, Protein Structure, Secondary, Protein Structure, Tertiary, Rubredoxins, Sequence Alignment, Structural Homology, Protein, Bacterial Proteins chemistry, Ferredoxins chemistry, Genomics, Pyrococcus furiosus chemistry
- Published
- 2004
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23. Neutron crystallographic study on rubredoxin from Pyrococcus furiosus by BIX-3, a single-crystal diffractometer for biomacromolecules.
- Author
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Kurihara K, Tanaka I, Chatake T, Adams MW, Jenney FE Jr, Moiseeva N, Bau R, and Niimura N
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- Deuterium Exchange Measurement, Hot Temperature, Hydrogen Bonding, Iron chemistry, Nitrogen chemistry, Protein Structure, Tertiary, Sulfur chemistry, Water chemistry, Neutron Diffraction instrumentation, Neutron Diffraction methods, Pyrococcus furiosus chemistry, Rubredoxins chemistry
- Abstract
The structure of a partially deuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, was determined by using the neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-3) at the JRR-3M reactor of the Japan Atomic Energy Research Institute. Data were collected at room temperature up to a resolution of 1.5 A, and the completeness factor of the data set was 81.9%. The model contains 306 H and 50 D atoms. A total of 37 hydration water molecules were identified, with 15 having all three atoms fully located and the remaining D2O molecules partially defined. The model has been refined to final agreement factors of R = 18.6% and Rfree = 21.7%. Several orientations of the O-D bonds of side chains, whose assignments from x-ray data were previously ambiguous, were clearly visible in the neutron structure. Although most backbone N-H bonds had undergone some degree of H/D exchange throughout the rubredoxin molecule, 5 H atom positions still had distinctly negative (H) peaks. The neutron Fourier maps clearly showed the details of an extensive set of H bonds involving the ND3+ terminus that may contribute to the unusual thermostability of this molecule.
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- 2004
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24. W3Y single mutant of rubredoxin from Pyrococcus furiosus: a preliminary time-of-flight neutron study.
- Author
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Li X, Langan P, Bau R, Tsyba I, Jenney FE Jr, Adams MW, and Schoenborn BP
- Subjects
- Crystallization, Deuterium, Neutron Diffraction, Point Mutation, Pyrococcus furiosus chemistry, Rubredoxins chemistry
- Abstract
Rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus maintains its native structure at high temperatures (373 K). In order to investigate the role of hydrogen bonding, hydration and chain dynamics in this thermostability, wavelength-resolved Laue neutron diffraction data have been collected from the W3Y single mutant (Trp3-->Tyr3) on the spallation neutron protein crystallography station (PCS) at Los Alamos Neutron Science Center. Data were measured at room temperature from nine crystal settings, each of approximately 12 h duration. The total data-measurement period was less than 5 d from a single crystal that had undergone H(2)O/D(2)O exchange. The nominal resolution of the data is 2.1 A.
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- 2004
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25. Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase.
- Author
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Clay MD, Cosper CA, Jenney FE Jr, Adams MW, and Johnson MK
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- Binding Sites, Electron Spin Resonance Spectroscopy methods, Spectrum Analysis, Raman, Substrate Specificity, Nitric Oxide metabolism, Oxidoreductases chemistry, Oxidoreductases metabolism, Pyrococcus furiosus enzymology
- Abstract
Nitric oxide (NO) has been used as a substrate analog to explore the structural and electronic determinants of enzymatic superoxide reduction at the mononuclear iron active site of Pyrococcus furiosus superoxide reductase (SOR) through the use of EPR, resonance Raman, Fourier transform IR, UV-visible absorption, and variable-temperature variable-field magnetic CD spectroscopies. The NO adduct of reduced SOR is shown to have a near-axial S = 32 ground state with ED = 0.06 and D = 12 +/- 2 cm(-1) (where D and E are the axial and rhombic zero-field splitting parameters, respectively) and the UV-visible absorption and magnetic CD spectra are dominated by an out-of-plane NO(-)(pi*)-to-Fe(3+)(dpi) charge-transfer transition, polarized along the zero-field splitting axis. Resonance Raman studies indicate that the NO adduct is six-coordinate with NO ligated in a bent conformation trans to the cysteinyl S, as evidenced by the identification of nu(N-O) at 1,721 cm(-1), nu(Fe-NO) at 475 cm(-1), and nu(Fe-S(Cys), at 291 cm(-1), via (34)S and (15)NO isotope shifts. The electronic and vibrational properties of the S = 32 (FeNO)(7) unit are rationalized in terms of a limiting formulation involving a high-spin (S = 52) Fe(3+) center antiferromagnetically coupled to a (S = 1) NO(-) anion, with a highly covalent Fe(3+)-NO(-) interaction. The results support a catalytic mechanism for SOR, with the first step involving oxidative addition of superoxide to form a ferric-peroxo intermediate, and indicate the important roles that the Fe spin state and the trans cysteinate ligand play in effecting superoxide reduction and peroxide release.
- Published
- 2003
- Full Text
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26. Resonance Raman characterization of the mononuclear iron active-site vibrations and putative electron transport pathways in Pyrococcus furiosus superoxide reductase.
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Clay MD, Jenney FE Jr, Noh HJ, Hagedoorn PL, Adams MW, and Johnson MK
- Subjects
- Binding Sites, Electron Transport, Mutagenesis, Site-Directed, Oxidoreductases genetics, Spectrum Analysis, Raman, Iron metabolism, Oxidoreductases metabolism, Pyrococcus furiosus enzymology
- Abstract
The resonance Raman spectrum of oxidized wild-type P. furiosus SOR at pH 7.5 and 10.5 has been investigated using excitation wavelengths between 406 and 676 nm, and vibrational modes have been assigned on the basis of isotope shifts resulting from global replacements of (32)S with (34)S, (14)N with (15)N, (56)Fe with (54)Fe, and exchange into a H(2)(18)O buffer. The results are interpreted in terms of the crystallographically defined active-site structure involving a six-coordinate mononuclear Fe center with four equatorial histidine ligands and axial cysteine and monodentate glutamate ligands (Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. C. (2000) Biochemistry 39, 2499-2508). Excitation into the intense (Cys)S(p(pi))-to-Fe(d(pi)) CT transition centered at 660 nm results in strong enhancement of modes at 298 cm(-1) and 323 cm(-1) that are assigned to extensively mixed cysteine S-C(beta)-C(alpha) bending and Fe-S(Cys) stretching modes, respectively. All other higher-energy vibrational modes are readily assigned to overtone or combination bands or to fundamentals corresponding to internal modes of the ligated cysteine. Weak enhancement of Fe-N(His) stretching modes is observed in the 200-250 cm(-1) region. The enhancement of internal cysteine modes and Fe-N(His) stretching modes are a consequence of a near-planar Fe-S-C(beta)-C(alpha)-N unit for the coordinated cysteine and significant (His)N(p(pi))-Fe(d(xy))-(Cys)S(p(pi)) orbital overlap, respectively, and have close parallels to type 1 copper proteins. By analogy with type 1 copper proteins, putative superexchange electron-transfer pathways to the mononuclear Fe active site are identified involving either the tyrosine and cysteine residues or the solvent-exposed deltaN histidine residue in a Y-C-X-X-H arrangement. Studies of wild-type at pH 10.5 and the E14A variant indicate that the resonance Raman spectrum is remarkably insensitive to changes in the ligand trans to cysteine and hence are inconclusive concerning the origin of the alkaline transition and the nature of sixth Fe ligand in the E14A variant.
- Published
- 2002
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27. Spectroscopic studies of Pyrococcus furiosus superoxide reductase: implications for active-site structures and the catalytic mechanism.
- Author
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Clay MD, Jenney FE Jr, Hagedoorn PL, George GN, Adams MW, and Johnson MK
- Subjects
- Binding Sites, Catalysis, Circular Dichroism, Electron Spin Resonance Spectroscopy, Oxidation-Reduction, Spectrophotometry, Ultraviolet, Structure-Activity Relationship, Oxidoreductases chemistry, Oxidoreductases metabolism, Pyrococcus furiosus enzymology
- Abstract
The combination of UV/visible/NIR absorption, CD and variable-temperature magnetic circular dichroism (VTMCD), EPR, and X-ray absorption (XAS) spectroscopies has been used to investigate the electronic and structural properties of the oxidized and reduced forms of Pyrococcus furiosus superoxide reductase (SOR) as a function of pH and exogenous ligand binding. XAS shows that the mononuclear ferric center in the oxidized enzyme is very susceptible to photoreduction in the X-ray beam. This observation facilitates interpretation of ground- and excited-state electronic properties and the EXAFS results for the oxidized enzyme in terms of the published X-ray crystallographic data (Yeh, A. P.; Hu, Y.; Jenney, F. E.; Adams, M. W. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508). In the oxidized state, the mononuclear ferric active site has octahedral coordination with four equatorial histidyl ligands and axial cysteinate and monodentate glutamate ligands. Fe EXAFS are best fit by one Fe-S at 2.36 A and five Fe-N/O at an average distance of 2.12 A. The EPR-determined spin Hamiltonian parameters for the high-spin (S = (5)/(2)) ferric site in the resting enzyme, D = -0.50 +/- 0.05 cm(-1) and E/D = 0.06, are consistent with tetragonally compressed octahedral coordination geometry. UV/visible absorption and VTMCD studies facilitate resolution and assignment of pi His --> Fe(3+)(t(2g)) and (Cys)S(p) --> Fe(3+)(t(2g)) charge-transfer transitions, and the polarizations deduced from MCD saturation magnetization studies indicate that the zero-field splitting (compression) axis corresponds to one of the axes with trans-histidyl ligands. EPR and VTMCD studies provide evidence of azide, ferrocyanide, hydroxide, and cyanide binding via displacement of the glutamate ligand. For azide, ferrocyanide, and hydroxide, ligand binding occurs with retention of the high-spin (S = 5/2) ground state (E/D = 0.27 and D < 0 for azide and ferrocyanide; E/D = 0.25 and D = +1.1 +/- 0.2 cm(-1) for hydroxide), whereas cyanide binding results in a low-spin (S = 1/2) species (g = 2.29, 2.25, 1.94). The ground-state and charge-transfer/ligand-field excited-state properties of the low-spin cyanide-bound derivative are shown to be consistent with a tetragonally elongated octahedral coordination with the elongation axis corresponding to an axis with trans-histidyl ligands. In the reduced state, the ferrous site of SOR is shown to have square-pyramidal coordination geometry in frozen solution with four equatorial histidines and one axial cysteine on the basis of XAS and UV and NIR VTMCD studies. Fe EXAFS are best fit by one Fe-S at 2.37 A and four Fe-N/O at an average distance of 2.15 A. VTMCD reveals a high-spin (S = 2) ferrous site with (Cys)S(p) --> Fe(2+) charge-transfer transitions in the UV region and (5)T(2g) --> (5)E(g) ligand-field transitions in the NIR region at 12400 and <5000 cm(-1). The ligand-field bands indicate square-pyramidal coordination geometry with 10Dq < 8700 cm(-1) and a large excited-state splitting, Delta (5)E(g) > 7400 cm(-1). Analysis of MCD saturation magnetization data leads to ground-state zero-field splitting parameters for the S = 2 ground state, D approximately +10 cm(-1) and E/D approximately 0.1, and complete assessment of ferrous d-orbital splitting. Azide binds weakly at the vacant coordination site of reduced SOR to give a coordination geometry intermediate between octahedral and square pyramidal with 10Dq = 9700 cm(-1) and Delta (5)E(g) = 4800 cm(-1). Cyanide binding results in an octahedral ferrous site with 10Dq = 10,900 cm(-1) and Delta (5)E(g) = 1750 cm(-1). The ability to bind exogenous ligands to both the ferrous and ferric sites of SOR is consistent with an inner-sphere catalytic mechanism involving superoxide binding at the ferrous site to yield a ferric-(hydro)peroxo intermediate. The structural and electronic properties of the SOR active site are discussed in relation to the role and bonding of the axial cysteine residue and the recent proposals for the catalytic mechanism.
- Published
- 2002
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28. Cluster-Dependent Charge-Transfer Dynamics in Iron–Sulfur Proteins
- Author
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Mao, Ziliang, Liou, Shu-Hao, Khadka, Nimesh, Jenney, Francis E, Goodin, David B, Seefeldt, Lance C, Adams, Michael WW, Cramer, Stephen P, and Larsen, Delmar S
- Subjects
Chemical Sciences ,Physical Chemistry ,Azotobacter vinelandii ,Bacteria ,Bacterial Proteins ,Catalytic Domain ,Electron Transport ,Ferredoxins ,Iron-Sulfur Proteins ,Models ,Molecular ,Oxidation-Reduction ,Oxidoreductases ,Protein Conformation ,Pseudomonas putida ,Pyrococcus furiosus ,Rubredoxins ,Medicinal and Biomolecular Chemistry ,Biochemistry and Cell Biology ,Medical Biochemistry and Metabolomics ,Biochemistry & Molecular Biology ,Biochemistry and cell biology ,Medical biochemistry and metabolomics ,Medicinal and biomolecular chemistry - Abstract
Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.
- Published
- 2018
29. Metalloproteins from Hyperthermophiles
- Author
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Jenney, Francis E., Jr., Adams, Michael W. W., and Horikoshi, Koki, editor
- Published
- 2011
- Full Text
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30. Characterization of the [3Fe–4S]0/1+ cluster from the D14C variant of Pyrococcus furiosus ferredoxin via combined NRVS and DFT analyses.
- Author
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Lauterbach, Lars, Gee, Leland B., Pelmenschikov, Vladimir, Jenney, Francis E., Kamali, Saeed, Yoda, Yoshitaka, Adams, Michael W. W., and Cramer, Stephen P.
- Subjects
PYROCOCCUS furiosus ,FERREDOXINS ,OXIDATION ,IRON-sulfur compounds ,SPECTROMETRY - Abstract
The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe–4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe–4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe–S clusters. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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31. Hydrogenesis in hyperthermophilic microorganisms: Implications for biofuels
- Author
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Chou, Chung-Jung, Jenney, Francis E., Adams, Michael W.W., and Kelly, Robert M.
- Subjects
- *
THERMOPHILIC microorganisms , *BIOMASS energy , *FERREDOXIN-NADP reductase , *ETHANOL as fuel , *HYDROGEN production , *BIOMASS production , *HYDROGENASE , *PHYSIOLOGY - Abstract
Abstract: Hydrothermal microbiotopes are characterized by the consumption and production of molecular hydrogen. Heterotrophic hyperthermophilic microorganisms (growth T opt⩾80°C) actively participate in the production of H2 in these environments through the fermentation of peptides and carbohydrates. Hyperthermophiles have been shown to approach the theoretical (Thauer) limit of 4mol of H2 produced per mole of glucose equivalent consumed, albeit at lower volumetric productivities than observed for mesophilic bacteria, especially enterics and clostridia. Potential advantages for biohydrogen production at elevated temperatures include fewer metabolic byproducts formed, absence of catabolic repression for growth on heterogeneous biomass substrates, and reduced loss of H2 through conversion to H2S and CH4 by mesophilic consortia containing sulfate reducers and methanogens. To fully exploit the use of these novel microorganisms and their constituent hydrogenases for biohydrogen production, development of versatile genetic systems and improvements in current understanding of electron flux from fermentable substrates to H2 in hyperthermophiles are needed. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
- View/download PDF
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