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E T Smith

Publications and source records attributed to E T Smith.

At least 37 records · Page 2Linked to original sources
PubMed1994

Solution 1H NMR determination of secondary structure for the three-iron form of ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S = 1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the beta-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded beta-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Identification of an unusual paramagnetic species and of three [2Fe-2S] clusters in the iron-only hydrogenase from the hyperthermophilic bacterium Thermotoga maritima.

Thermotoga maritima is a H2-producing, fermentative anaerobe and is one of the most thermophilic bacteria known. Its iron-only (Fe-)hydrogenase was previously shown to be a homotetramer and to contain two [4Fe-4S] and two [2Fe-2S] clusters per monomer, but the enzyme lacked the characteristic EPR signal of the oxidized H cluster, the proposed site of H2 catalysis in mesophilic Fe-hydrogenases (Juszczak, A., Aono, S. and Adams, M.W.W. (1991) J. Biol. Chem. 266, 13834-13841). The two types of cluster were shown by spectroelectrochemistry to have reduction potentials (Em) of -390 and -440 mV, respectively. We have now identified two additional redox centers in the enzyme, a [2Fe-2S] center with a higher reduction potential (Em = -365 mV) and an unusual paramagnetic species (Em > -200 mV). The higher potential [2Fe-2S] center can be reduced by sodium dithionite at pH 6.0 and exhibits an axial-type EPR signal with gz = 2.026 and gy = gx = 1.940. The two lower potential [2Fe-2S] centers are fully reduced by sodium dithionite only at pH 10.0. Both of these clusters in their reduced states exhibit rhombic-type EPR signals with gz = 2.005, gy = 1.955, and gx = 1.921. This hydrogenase is therefore thought to contain three [2Fe-2S] clusters, as well as two [4Fe-4S] clusters. In addition, a nearly isotropic EPR signal (g = 2.01) was observed when the enzyme was anaerobically oxidized by organic dyes such as thionine (E alpha = 64 mV) or 2,6-dichlorophenolindophenol (E alpha = 217 mV). This resonance was not observed at 20 K due to relaxation broadening and therefore did not arise from a conventional organic radical. The oxidized enzyme was fully active in an H2 production assay, and also reacted directly with H2. In contrast, the air-oxidized enzyme was inactive and did not exhibit the g = 2.01 EPR signal. This resonance was assigned to a novel paramagnetic species with an approximate Em value of -70 mV. It is thought to be associated with the H2 activating site of this atypical Fe-hydrogenase.

Bacterial Proteins↗
PubMed1994

Pyruvate ferredoxin oxidoreductases of the hyperthermophilic archaeon, Pyrococcus furiosus, and the hyperthermophilic bacterium, Thermotoga maritima, have different catalytic mechanisms.

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from two hyperthermophiles, the archaeon Pyrococcus furiosus (Pf, Topt = 100 degrees C) and the bacterium Thermotoga maritima (Tm, Topt = 80 degrees C). Each catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 near the optimal growth temperature of the organism and are virtually inactive at 25 degrees C. Both PORs contain a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] ferredoxin-type clusters. We have now shown, using EPR spectroscopy and metal analyses, that PfPOR also contains an unusual copper center that is not present in Tm POR. In addition, distinct catalytic intermediates were generated in both enzymes by the addition, separately and in combination, of the substrates pyruvate and CoASH, and these were examined by EPR spectroscopy. The addition of pyruvate to oxidized Pf POR produced an isotropic signal centered at g = 2.01, which was measurably broader in the presence of pyruvate-2(13)C. This signal, which was assigned to a (hydroxyethyl)thiamine pyrophosphate radical intermediate, was not observed in Tm POR under the same experimental conditions. Incubation of the oxidized enzymes with CoASH resulted in the partial reduction of the copper site in Pf POR and the partial reduction of a novel iron-sulfur center in Tm POR, which was not seen in the dithionite-reduced enzyme. The addition of both pyruvate and CoASH to the PORs in their oxidized states resulted in the reduction of the same iron-sulfur centers that are reduced by sodium dithionite.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaea↗
PubMed1994

Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster.

Amino Acid Sequence↗
PubMed1994

Comparison of magnetic resonance angiography, duplex ultrasound, and digital subtraction angiography in assessment of extracranial internal carotid artery stenosis.

The results of a prospective study comparing ultrasound, intra-arterial digital subtraction angiography, and magnetic resonance angiography in the assessment of the degree of extracranial internal carotid artery stenosis are reported in patients with symptoms of recent carotid territory ischaemia. A total of 70 patients and 137 vessels were examined by all three techniques. The results obtained by each technique were reported blind. The mean difference (SD) for the comparison of magnetic resonance angiography and digital subtraction angiography was -0.7 (14)%, for ultrasound and digital subtraction angiography 3.1 (15)%, and for magnetic resonance angiography and ultrasound -3.8 (15)%. The level of agreement was greater for the more tightly stenosed vessels. With the assumption that the results of the digital subtraction angiogram reflect the true situation, the sensitivity and specificity in the detection of > or = 30% stenoses were 93% and 82% with ultrasound and 89% and 82% with magnetic resonance angiography; for stenoses > or = 70% 93% and 92% with ultrasound and 90% and 95% with magnetic resonance angiography; and for stenoses of 70-99% 89% and 93% with ultrasound and 86% and 93% with magnetic resonance angiography. For occlusion the values were 93% and 99% with ultrasound and 80% and 99% with magnetic resonance angiography. Increased sensitivity and specificity were obtained when analysis was confined to those vessels in which ultrasound and magnetic resonance angiography were in agreement over classification. It is thus possible to accurately categorize the degree of stenosis of the extracranial internal carotid artery from a combination of ultrasound and magnetic resonance angiography. The adoption of this combination for the investigation of patients before carotid endarterectomy removes the risk associated with conventional angiography and represents an important advance in the management of carotid stenosis.

Adult↗
PubMed1993

Medium-chain acylcoenzyme-A dehydrogenase deficiency. Not just another Reye syndrome.

The medium-chain acylcoenzyme-A dehydrogenase enzyme (MCAD) is involved in the initial stages of breakdown of medium-chain-length fatty acids in mitochondria. A deficiency of this enzyme can become symptomatic during a catabolic state and may resemble Reye syndrome. We report the case of a 17-month-old white girl whose 1-day history of vomiting and agitation followed by generalized unresponsiveness caused her to be brought to a local emergency department. She died after resuscitative efforts failed, and an autopsy revealed severe cerebral edema, marked hepatic steatosis, and steatosis of the renal tubule epithelia. Electron microscopy of the liver showed cytoplasmic lipid spherules and mitochondria with dense matrices, changes similar to Reye syndrome; however, postmortem examination of the decedent's blood revealed elevated medium-chain-length acylcarnitines diagnostic of an MCAD deficiency. Although uncommon, a deficiency of this enzyme should be considered in apparent Reye syndrome victims.

Acyl-CoA Dehydrogenase↗
PubMed1992

Proton NMR investigation of the oxidized three-iron clusters in the ferredoxins from the hyperthermophilic archae Pyrococcus furiosus and Thermococcus litoralis.

The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.

Amino Acid Sequence↗
PubMed1992

A temperature-controlled, anaerobic cell for direct electrochemical studies.

The construction and operation of a cell for temperature-controlled, direct electrochemical studies of oxygen-sensitive materials are described. The borosilicate cell contains a pyrolytic graphite working electrode, a Ag/AgCl reference electrode, and a platinum counter electrode, all of which can be readily interchanged with other types of electrodes. It is surrounded by a water jacket constructed of steel and Lexan, which can easily maintain temperatures between 4 degrees C and at least 90 degrees C. The entire cell was designed to minimize the number, complexity, and expense of components, as well as minimize required sample volume (250 microliters) and sources of oxygen leakage. As examples of the cell's utility, the redox properties of two common organic redox dyes, methyl viologen and thionin, were determined by differential pulse voltammetry at temperatures from 30 to 90 degrees C.

Anaerobiosis↗
PubMed1992

Direct electrochemical studies of hydrogenase and CO dehydrogenase.

The reduction potentials of two relatively high-molecular-mass enzymes were determined directly at an edge pyrolytic graphite electrode by using square-wave voltammetry. The equilibrium reduction potential versus standard hydrogen electrode was determined for Clostridium pasteurianum hydrogenase I (E'0 = -377 +/- 10 mV; molecular mass 60 kDa) and Rhodospirillum rubrum carbon monoxide dehydrogenase (E'0 = -418 +/- 7 mV; molecular mass 62 kDa). The reduction potential of each enzyme was pH-independent, and one electron was transferred per redox centre. The reduction potential was determined to be identical for the CO dehydrogenase, semi-apo-(CO dehydrogenase), and CO dehydrogenase with carbonyl sulphide (COS) or cyanide bound. The electron-transferring efficiency of CO dehydrogenase was affected by two inhibitors, COS and cyanide, as indicated by a diminished analytic current.

Aldehyde Oxidoreductases↗
PubMed1991

A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.

The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.

Amino Acid Sequence↗
PubMed1990

Redox properties of several bacterial ferredoxins using square wave voltammetry.

The equilibrium reduction potential of the 2[4Fe-4S] ferredoxin (Fd) isolated from four different bacterial strains was determined at a methyl viologen-modified gold electrode using square wave voltammetry. The observed reduction potential at pH 8 for Clostridium thermoaceticum Fd was -385 mV; Clostridium pasteurianum, -393 mV; Clostridium thermosaccharolyticum, -408 mV; and Chromatium vinosum, -460 mV versus normal hydrogen electrode at 25 degrees C. The reduction potential of the C. pasteurianum Fd was found to be pH independent from pH 6.4 to 8.7, indicating that the electron transfer mechanism does not involve proton exchange. In contrast, the reduction potential of the C. thermosaccharolyticum Fd was found to be pH dependent from pH 6.4 to 8.7, with pKox approximately 7 and pKred approximately 7.5. The +30 mV change in reduction potential from pH 8.7 to 6.4 was attributed to an electrostatic interaction between the iron-sulfur cluster II and the protonated histidine 2 residue located about 6 A away. The Ch. vinosum Fd interacted reversibly at the methyl viologen-modified gold electrode, and its reduction potential was verified using visible spectroelectrochemistry. The reduction potential of Ch. vinosum Fd was found to be 30 mV more positive than previously reported. The similarities of the bacterial Fd reduction potentials are discussed in terms of the homology of their primary structure as reflected by the similarities in the visible and circular dichroic spectra.

Chromatium↗
PubMed1986

A method for identification of vertebral level.

A method of spinal level marking applicable particularly for use in thoracolumbar posterior spinal operations is described. The use of patent blue V dye in this procedure is discussed in a consecutive series of over 100 cases. No serious adverse effects were observed. The technique ensures accurate identification of spinal marking and helps to minimize anaesthetic time.

Coloring Agents↗
PubMed1977

A technique for closure of the lesser curvature of the stomach used in the von Eiselberg-Hofmeister reconstruction after subtotal gastrectomy.

A technique for closing the lesser curvature of the stomach after subtotal gastrectomy has been used in 847 subtotal gastrectomies. In this group, one patient had transient postoperative bleeding and none had anastomotic leakage. The technique is recommended as a simple method of obtaining a secure and hemostatic closure which can be accomplished rapidly and which does not require the use of special instruments or material.

Gastrectomy↗
PubMed1976

The lethal hit stage of mouse T and non-T cell-mediated cytolysis: differences in cation requirements and characterization of an analytical "cation pulse" method.

We investigated in detail the cation requirements of two different systems of mouse cell-mediated cytolysis in vitro, at their recognition, post-recognition hit and target cell disintegration stages. In T cell-mediated cytolysis, respectively Mg++ or Ca++, Ca++, and no cations, were required. In non-T cell-mediated hemolysis, respectively no cations, Mg++, and no cations, were required. Two main conclusions can be drawn. First, the cation requirements are different from one system to the other especially at the post-recognition hit stage, which strongly suggests the existence of differences between both systems as to the actual mechanism of lysis. Second, the cation requirements are different within each system from one step to another. This formed the basis of a step-by-step analysis of the lytic process, leading to the characterization of a cation pulse method for the experimental isolation and further study of the post-recognition hit stage of cell-mediated cytolysis.

Animals↗