PubMed Health⌕ Search

Biomedical subjects

L E Mortenson

Publications and source records attributed to L E Mortenson.

At least 55 records · Page 3Linked to original sources
PubMed1985

The iron-sulfur cluster composition of Escherichia coli nitrate reductase.

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.

Circular Dichroism↗
PubMed1985

Anaerobic multiphasic gel electrophoresis of the molybdoproteins in extracts of Clostridium pasteurianum.

Simple procedures using multiphasic buffer systems for anaerobic electrophoresis have been devised to identify oxygen-labile metalloproteins. An anaerobic slab gel apparatus was developed with cooling and design for anaerobic conditions. Included is a procedure to remove sample wells after stacking proteins in a crude extract, to prevent streaking (background) caused by continuous leakage of "nonstacked protein" from the sample wells. Identification of eleven Mo zones in extracts of Clostridium pasteurianum demonstrates the usefulness of the technique in identifying radiolabeled oxygen-labile proteins in cell-free crude lysates.

Anaerobiosis↗
PubMed1985

Formate dehydrogenase molybdenum and tungsten sites--observation by EXAFS of structural differences.

Preliminary EXAFS data has been collected on the molybdenum (K-edge) in C. pasteurianum formate dehydrogenase and the tungsten (LIII-edge) in C. thermoaceticum formate dehydrogenase. In the presence of dithionite, the tungsten enzyme was devoid of W = O bonds, and exhibited average W-(O, N) and W-S bond lengths of 2.13 +/- 0.03 A and 2.39 +/- 0.03 A, respectively. In sharp contrast, the C. pasteurianum molybdenum site has three Mo = O bonds with an average bond length of 1.74 +/- 0.03 A. It is also the first molybdenum enzyme found lacking Mo-S bonds, and does not appear to be redox active in the presence of formate or dithionite. Model compounds WO2(8-hydroxyquinoline)2 = WO2(ox)2, and WO2(8 mercaptoquinoline)2 = WO2(tox)2, were also examined. Respective predicted bond lengths for WO2(ox)2 and WO2(tox)2 were W = O of 1.71, 1.73 A; W-N of 2.31, 2.29 A; W-O or W-S of 1.92 or 2.40 A, with estimated uncertainties of +/- 0.03 A.

Aldehyde Oxidoreductases↗
PubMed1985

Identification of molybdoproteins in Clostridium pasteurianum.

Cells of Clostridium pasteurianum whose N source is switched from NH3 to N2 accumulate large amounts of molybdenum beginning 1.5 h before the detection of nitrogenase activity. Anaerobic multiphasic gel electrophoresis and anion-exchange chromatography were used to identify the molybdoproteins and molybdenum-containing components present in N2-fixing cells. In addition to molybdate, six distinct 99Mo-labeled species were detected, i.e., a membrane fragment, the MoFe protein of nitrogenase, formate dehydrogenase, a Mo "binding-storage" protein, a 30-kilodalton molybdoprotein, and a low-molecular-weight molybdenum species. Of these, the MoFe protein, formate dehydrogenase, and the Mo binding-storage protein were present in more than one zone because of complex formation with other proteins, partial denaturation, and variation in the amount of Mo bound to the protein, respectively. In addition to the six proteins, a soluble "free" Mo cofactor in the cytosol was detected by showing that it reconstituted nitrate reductase activity in crude extracts of the Neurospora crassa mutant nit-1.

Anaerobiosis↗
PubMed1985

Regulation and order of involvement of molybdoproteins during synthesis of molybdoenzymes in Clostridium pasteurianum.

The accumulation of 99Mo (from 99MoO4(2-) into molybdenum-containing species in Clostridium pasteurianum was investigated to identify the molybdoprotein(s) involved in Mo metabolism. Mo accumulation by clostridial cells during the derepression of the nitrogenase system increased substantially beginning 1.5 h before nitrogenase activity was detected. The increase in Mo accumulation by the cells is a result of the incorporation of Mo into a high-molecular-weight molybdenum species (suspected membrane fragments), a low-molecular-weight molybdenum species, a Mo binding-storage protein, a 30-kilodalton molybdoprotein, and formate dehydrogenase. Mo incorporation into the MoFe protein was detected 1 h after the onset of metal uptake. Kinetics of Mo accumulation into the molybdoproteins during the derepression of nitrogenase suggests that Mo incorporation or uptake or both occur in the following sequence: (i) membranes and MoO4(2-), (ii) a low-molecular-weight molybdenum species, (iii) Mo binding-storage protein and a 30-kilodalton molybdoprotein, (iv) formate dehydrogenase, and (v) the MoFe protein. The intracellular level of all molybdenum components except the MoFe protein appears to be influenced by the availability of Mo. Clostridial cells grown in the presence of a limiting amount of Mo became Mo deficient as a result of growth and a MoO4(2-) supplement added to such cells rapidly accumulated within the cells to levels five times that found in steady-state nitrogen-fixing cells. The Mo accumulated by the Mo-deficient cells was rapidly incorporated into preformed demolybdoproteins in the absence of de novo protein synthesis. The increase in Mo accumulation by Mo-deficient cells was a result of an increase in all molybdoproteins except the MoFe protein.

Bacterial Proteins↗
PubMed1984

Mössbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5.

The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides. We have studied oxidized samples of the enzyme with Mössbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01. The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances. One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz. These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. The Mössbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms. Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated [4Fe-4S] cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a [4Fe-4S] core. The Mössbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type [4Fe-4S] clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme.

Clostridium↗
PubMed1984

The physical and catalytic properties of hydrogenase II of Clostridium pasteurianum. A comparison with hydrogenase I.

Hydrogenase II of Clostridium pasteurianum is a monomeric protein of Mr = 53,000 containing 8 iron and 8 acid-labile sulfide atoms/mol. It is distinct from hydrogenase I from the same organism (Mr = 60,000 12 Fe and 12 S2-/mol). Metal analyses showed that neither hydrogenase contains nickel or any other metals in significant amounts. The iron atoms of hydrogenase II resisted chelation by 2,2'-bipyridyl but all were susceptible when the enzyme was treated with ferricyanide. Core extrusion indicated the presence of two [4Fe-4S] clusters in hydrogenase II and EPR spectroscopy showed two distinct paramagnetic species which could be interpreted as one [4Fe-4S]2+(2+,1+) and one [4Fe-4S]2+(2+,3+) per molecule. The absorption coefficient of H2-reduced hydrogenase II at 420 nm was 23,000 M-1 cm-1 with a A420 / A275 ratio of 0.27. There were large differences between hydrogenase I and hydrogenase II in the absorption spectra of the air-oxidized, H2-reduced, and dithionite-reduced forms of the enzymes. Hydrogenase II catalyzed H2 evolution with methyl viologen or ferredoxin as the electron carrier, and H2 oxidation with methylene blue or methyl viologen as the electron acceptor. Apparent Km values were determined for all these reactions with both hydrogenases. Hydrogenase II is a relatively inactive enzyme, except in the reduction of methylene blue by H2. The pH dependencies of H2 oxidation were similar for both hydrogenases but were very different in H2 evolution. The activation energy values were much higher for H2 catalysis by hydrogenase II than for hydrogenase I. The two hydrogenases have the same sensitivity to inactivation by O2 but differ in their sensitivity to metal-chelating reagents and to CO. Hydrogenase I is more readily inhibited by CO but hydrogenase II binds CO irreversibly. From the above data, a mechanism is proposed to account for the observed differences in the catalytic activities of hydrogenase I and hydrogenase II.

Binding Sites↗
PubMed1984

Formate dehydrogenase of Clostridium pasteurianum.

Formate dehydrogenase was purified to electrophoretic homogeneity from N2-fixing cells of Clostridium pasteurianum W5. The purified enzyme has a minimal Mr of 117,000 with two nonidentical subunits with molecular weights of 76,000 and 34,000, respectively. It contains 2 mol of molybdenum, 24 mol of nonheme iron, and 28 mol of acid-labile sulfide per mol of enzyme; no other metal ions were detected. Analysis of its iron-sulfur centers by ligand exchange techniques showed that 20 iron atoms of formate dehydrogenase can be extruded as Fe4S4 centers. Fluorescence analysis of its isolated molybdenum centers suggests it is a molybdopterin. The clostridial formate dehydrogenase has a pH optimum between 8.3 and 8.5 and a temperature optimum of 52 degrees C. The Km for formate is 1.72 mM with a Vmax of 551 mumol of methyl viologen reduced per min per mg of protein. Sodium azide competes competitively with formate (K1 = 3.57 microM), whereas the inactivation by cyanide follows pseudo-first-order kinetics with K = 5 X 10(2) M-1 s-1.

Aldehyde Oxidoreductases↗
PubMed1983

Comparison of data elements in guidelines developed independently in community hospital oncology programs.

In summary we would like to thank the communities that participated in this study. A comparison of data elements in the guidelines developed by these communities demonstrated similarity in areas of diagnosis, pretreatment evaluation and staging. In the area of treatment it was apparent that physicians writing these guidelines were less autocratic in that more flexibility, more options and less specificity in recommendations was found. That this is not necessarily unfavorable is reflected in the fact that variations existed where the cancer literature is unsettled.

Community Health Services↗
PubMed1982

Resonance Raman and electron paramagnetic resonance studies on oxidized and ferricyanide-treated Clostridium pasteurianum ferredoxin. Vibrational assignments from 34S shifts and evidence for conversion of 4 to 3 iron-sulfur clusters via oxidative damage. Vibrational assignments from 34S shifts and evidence for conversion of 4 to 3 iron-sulfur clusters via oxidative damage.

Resonance Raman spectra are reported for oxidized ferredoxin from Clostridium pasteurianum and for protein reconstituted with 34S2-, using 4579 A laser excitation. The spectra are of much higher quality than that previously reported, and the 34S shifts provide assignments of the Fe-S modes. After treatment with ferricyanide, the resonance Raman spectrum closely resembles that of the [3Fe-3S] protein, ferredoxin II from D. gigas; the 34S shifts aid in assignments of the [3Fe-3S] modes. The epr signal associated with the [3Fe-3S] cluster (g = 2.01) corresponds maximally to 0.80 spin/molecule. Anaerobic addition of excess sulfide to the reduced, ferricyanide-treated protein regenerates a [4Fe-4S]1+ epr spectrum, equivalent in intensity to the [3Fe-3S] signal. The ubiquitous occurrence of a g = 2.01 signal in preparations of [4Fe-4S] proteins can be attributed to variable amounts of [3Fe-3S] cluster generated by adventitious oxidation. The ready conversion of [4Fe-4S] to [3Fe-3S] clusters in C. pasterianum raises the possibility that some [3Fe-3S] proteins may actually arise by oxidative damage of [4Fe-4S] proteins during aerobic isolation.

Clostridium↗