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M W Adams

Publications and source records attributed to M W Adams.

At least 145 records · Page 8Linked to original sources
PubMed1989

Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus.

The archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative type metabolism in which H2 and CO2 are the only detectable products. The organism also reduces elemental sulfur (S0) to H2S. Cells grown in the absence of S0 contain a single hydrogenase, located in the cytoplasm, which has been purified 350-fold to apparent homogeneity. The yield of H2 evolution activity from reduced methyl viologen at 80 degrees C was 40%. The hydrogenase has a Mr value of 185,000 +/- 15,000 and is composed of three subunits of Mr 46,000 (alpha), 27,000 (beta), and 24,000 (gamma). The enzyme contains 31 +/- 3 g atoms of iron, 24 +/- 4 g atoms of acid-labile sulfide, and 0.98 +/- 0.05 g atoms of nickel/185,000 g of protein. The H2-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 15 K, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by O2 and is remarkably thermostable. Most of its H2 evolution activity is retained after a 1-h incubation at 100 degrees C. Reduced ferredoxin from P. furiosus also acts as an electron donor to the enzyme, and a 350-fold increase in the rate of H2 evolution is observed between 45 and 90 degrees C. The hydrogenase also catalyzes H2 oxidation with methyl viologen or methylene blue as the electron acceptor. The temperature optimum for both H2 oxidation and H2 evolution is greater than 95 degrees C. Arrhenius plots show two transition points at approximately 60 and approximately 80 degrees C independent of the mode of assay. That occurring at 80 degrees C is associated with a dramatic increase in H2 production activity. The enzyme preferentially catalyzes H2 production at all temperatures examined and appears to represent a new type of "evolution" hydrogenase.

Archaea↗
PubMed1989

Electron paramagnetic resonance studies of the low temperature photolytic behavior of oxidized hydrogenase I from Clostridium pasteurianum.

The effects of CO and O2 on the EPR spectrum of oxidized Clostridium pasteurianum hydrogenase I have been investigated both before and after prolonged exposure to white light at 8 K and 30 K. Low concentrations of O2 were found to induce analogous changes in the EPR spectrum as CO, i.e. conversion of the rhombic signal with g approximately 2.10, 2.04, 2.00, a characteristic of the novel H2-activating center in oxidized Fe-hydrogenases, to an axial signal with g approximately 2.07, 2.01, 2.01. The results suggest a common binding site and mode of coordination for CO and O2 and permit rationalization of conflicting reports from different laboratories concerning the EPR properties of oxidized Fe-hydrogenases. The CO- and O2-induced axial EPR signals were found to be light-sensitive at low temperatures. Moreover, they exhibited indistinguishable and unusual photolysis behavior with the dominant photo-product being dependent on the temperature at which illumination was performed. At 8 K, photodissociation of CO or O2 occurs, resulting in an EPR signal identical with that of the oxidized enzyme in the absence of CO or O2. However, at 30 K, the dominant photoproduct is a rhombic EPR signal with g approximately 2.26, 2.12, 1.89. While the origin of this new EPR signal is uncertain, the g-value anisotropy and relaxation characteristics resemble those of a low spin Fe(III) center. These two photoproducts cannot be thermally or photolytically interconverted, but both are quantitatively reconverted to the original axial EPR signal on warming in the dark to 200 K. A tentative working hypothesis for the nature of the H2-activating center of Fe-hydrogenases is presented that is consistent with the available physiochemical data and permits rationalization of the novel photolysis behavior.

Carbon Monoxide↗
PubMed1989

Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

The iron and acid-labile sulfide contents and the electron paramagnetic resonance (EPR) properties of hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum (strain W5) have been determined on the basis of quantitative amino acid analyses. The iron and acid-labile sulfide values are approximately 20 and 18 atoms per molecule of hydrogenase I and 14 and 11 atoms per molecule of hydrogenase II, respectively. These amounts are substantially greater than previously reported values, which relied on protein concentration determined by colorimetric assay. The oxidized hydrogenases exhibit unusual EPR signals that originate from a novel type of iron-sulfur center, termed the hydrogenase or H cluster, which covalently binds the inhibitor CO. This EPR signal represents approximately one unpaired electron per molecule in each enzyme with and without bound CO, which is consistent with the presence of one oxidized H cluster (S = 1/2) per enzyme molecule. The two enzymes also contain ferredoxin-type four-iron centers or F clusters. The EPR signals from the F clusters observed in the reduced forms of hydrogenase I and hydrogenase II account for approximately four and one unpaired electron per molecule, respectively. We conclude from the iron determinations and the EPR results, together with a reevaluation of previous spectroscopic data, that in both hydrogenases the H cluster probably comprises six iron atoms. Mechanistic models of the two hydrogenases are presented that account for their cluster compositions and the dramatic differences in their catalytic activities.

Amino Acids↗
PubMed1989

A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.

Amino Acids↗
PubMed1988

Effects of variety and particle size upon utilization of fescue hay by lactating dairy cows and dairy heifers.

Missouri-96 and Kentucky-31 hays were chopped with a tub grinder containing screens with apertures of 31, 63, or 100 mm in diameter and fed to dairy cows or heifers. Particle sizes (geometric mean diameter) were 1218, 1486, and 1933 micron, respectively, for the 31, 63, and 100-mm treatments. In Trial 1, the six treatments were fed ad libitum to 24 lactating cows; concentrate was offered at 1 kg/2 kg of milk. Dry matter intake and NDF digestibility were greater for Kentucky-31, but there were no other effects of variety. Particle size did not affect DM intake, DM or fiber digestibility, nitrogen partition, milk yield, or milk fat percentage. Crude protein digestibility was greatest for the 63-mm particle size treatment. In trial 2, the six treatments were fed ad libitum to 24 dairy heifers. Intake was greater for the 31 than for the 100-mm treatment but was unaffected by variety. Variety and particle size did not affect DM digestibility, nitrogen utilization, or daily gain. In this study fescue hay chopped through screens having apertures ranging from 31 to 100 mm was without effect on milk yield or composition. Reducing particle size increased intake and nitrogen utilization but not DM or fiber digestibility.

Animal Feed↗
PubMed1987

The mechanisms of H2 activation and CO binding by hydrogenase I and hydrogenase II of Clostridium pasteurianum.

Hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum have been investigated by electron paramagnetic resonance (EPR) spectroscopy, in the presence and absence of the inhibitor, CO. These hydrogenases contain both a novel type of iron-sulfur cluster (H), which is the proposed site of H2 catalysis, and ferredoxin-type [4Fe-4S] clusters (F). The results show that the H clusters of these two hydrogenases have very different properties. The H cluster of oxidized hydrogenase II (Hox-II) exhibits three distinct EPR signals, two of which are pH-dependent. Hox-II binds CO reversibly to give a single, pH-independent species with a novel, rhombic EPR spectrum. The H cluster of reduced hydrogenase II (Hred-II) does not react with CO. In contrast, the EPR spectrum of Hox-I appears homogeneous and independent of pH. Hox-I has a much lower affinity for CO than Hox-II, and binds CO irreversibly to give an axial EPR signal. Hred-I also binds CO irreversibly. The EPR spectra of Fred-I and Fred-II show little or no change after CO treatment. Prior exposure to CO does not affect the catalytic activity of the reduced or oxidized hydrogenases when assayed in the absence of CO, but both enzymes are irreversibly inactivated if CO is present during catalysis. Mechanisms for H2 activation by hydrogenase I and hydrogenase II are proposed from the determined midpoint potentials (Em, pH 8.0) of H-I and H-II (Em approximately -400 mV, -CO; approximately -360 mV, +CO), F-I (Em = -420 mV, +/- CO), and F-II (Em = -180 mV, +/- CO). These allow one to rationalize the different modes of CO binding to the two hydrogenases and suggest why hydrogenase II preferentially catalyzes H2 oxidation. The results are discussed in light of recent spectroscopic data on the structures of the two H clusters.

Carbon Monoxide↗
PubMed1987

An investigation of hydrogenase I and hydrogenase II from Clostridium pasteurianum by resonance Raman spectroscopy. Evidence for a [2Fe-2S] cluster in hydrogenase I.

Resonance Raman spectra are reported for hydrogenase I and II from Clostridium pasteurianum. These spectra show overlapping bands with contributions from [4Fe-4S] clusters, known to be present in these enzymes, and from novel FeS centers of hitherto undefined structure. For hydrogenase I there are strong bands at 288 and 394 cm-1, which are seen in [2Fe-2S] proteins and in no other FeS species so far examined. In contrast these bands do not appear for hydrogenase II, whose resonance Raman spectrum is dominated by [4Fe-4S] cluster modes. These results provide the first structural information on the hydrogenase I FeS center involved in H2 activation and demonstrate structural differences between hydrogenase I and hydrogenase II.

Clostridium↗
PubMed1987

EPR and electron nuclear double resonance investigation of oxidized hydrogenase II (uptake) from Clostridium pasteurianum W5. Effects of carbon monoxide binding.

Two different hydrogenases have been isolated from Clostridium pasteurianum W5. Hydrogenase II (uptake) is active in H2 oxidation while hydrogenase I (bidirectional) is active both in H2 oxidation and evolution. Previous EPR and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I have now been complemented by analogous studies on oxidized 57Fe-enriched hydrogenase II and its CO derivative (using 12CO and 13CO). Binding of CO greatly changes the EPR spectrum of oxidized hydrogenase II, and use of 13CO leads to resolved hyperfine splitting from interaction with a single 13CO molecule (AC approximately 34 MHz). This coupling is over 50% larger than that seen for hydrogenase I. 57Fe ENDOR disclosed two types of iron site in both oxidized hydrogenase II and its CO derivative. Combination of EPR, ENDOR, and Mössbauer results shows that site 1 has AFe1 = 18 MHz shifting to approximately 30 MHz upon CO binding and consisting of two Fe atoms and site 2 has A2 approximately 7 MHz shifting to approximately 10 MHz and containing a single Fe. These results are very similar to those seen for hydrogenase I, which indicates that a structurally similar 3Fe cluster, believed to be the catalytically active site, is present in both. Proton ENDOR shows a solvent exchangeable resonance only in the CO derivative of hydrogenase II. This indicates a structural difference between hydrogenases I and II that is brought out by CO binding. No evidence of 14N coordination to the cluster is seen for either enzyme.

Carbon Monoxide↗
PubMed1987

Mössbauer study of Clostridium pasteurianum hydrogenase II. Evidence for a novel three-iron cluster.

Hydrogenase II contains two iron-sulfur clusters, one of the [4Fe-4S] type and one of unknown structure with unusual spectral properties (H-cluster). Using Mössbauer spectroscopy we have studied the H-cluster under a variety of conditions. In the reduced state the cluster exhibits, in zero magnetic field, spectra with the typical 2:1 quadrupole pattern of reduced [3Fe-4S] clusters. However, whereas the latter are paramagnetic (S = 2) the H-cluster is diamagnetic (S = 0). Upon oxidation and exposure to CO the H-cluster exhibits an S = 1/2 EPR spectrum with g values at 2.03, 2.02, and 2.00. In this state, the Mössbauer spectra reveal two cluster subsites with magnetic hyperfine coupling constants AI = +26.5 MHz and AII = -30 MHz. ENDOR data obtained by Hoffman and co-workers (Telser, J., Benecky, M. J., Adams, M. W. W., Mortenson, L. E., and Hoffman, B. M. (1986) J. Biol. Chem. 261, 13536-13541) show a 57Fe resonance at AIII approximately equal to 9.5 MHz. Analysis of the Mössbauer spectra shows that this resonance represents one iron site. Our studies of the reduced and CO-bound oxidized states of hydrogenase II suggest that the H-cluster contains three iron atoms. The data obtained for the oxidized H-cluster suggest a novel type of 3-Fe cluster and bear little resemblance to those reported for oxidized [3Fe-4S] clusters with g = 2.01 EPR signals. In the reduced sample the [4Fe-4S]1+ cluster appears to occur in a mixture of two distinct electronic states.

Clostridium↗
PubMed1986

An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5.

Previous Mössbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel Mössbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters. The observation of 57Fe ENDOR signals from two types of iron site ([A1c] approximately 30-34 MHz; [A2c] approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by [A1c] confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants.

Carbon Monoxide↗
PubMed1986

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases.

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, Mössbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.

Anaerobiosis↗
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↗
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↗
PubMed1980

Biological activity of synthetic molybdenum-iron-sulphur, iron-sulphur and iron-selenium analogues of ferredoxin-type centres.

The molybdenum-iron-sulphur cluster [Fe6Mo2S8(SCH2CH2OH)9]3-, which contains two Fe3MoS4 cubane-like centres, is the best plausible analogue available to date for the molybdenum site of the nitrogenase enzymes. The iron-sulphur cluster [Fe4S4(S . CH2CH2OH)4]2- and the iron-selenium cluster [Fe4Se4(S . CH2CH2OH)4]2- are structural analogues of the ferredoxin Fe4S4 active centre. All three clusters would replace ferredoxin and mediate electron transfer to Clostridium pasteurianum hydrogenase in a H2-evolving system with sodium dithionite as the electron donor. The clusters would not replace hydrogenases which themselves are unable to evolve H2 from reduced ferredoxins. The molybdenum-iron-sulphur cluster would also replace ferredoxin in a chloroplast-ferredoxin-hydrogenase H2 evolving system.

Binding Sites↗
PubMed1980

Hydrogenase.

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Aerobiosis↗