Clostridium Pasteurianum W5 Research Articles

Super reduced Fe4S4 cluster of Balch's dithiolene series

A super reduced Fe(4)S(4) cluster with a sulfur based radical, [NBu(4)](4)[Fe(3)(III)Fe(II)(μ(3)-S)(4)(mnt)(3)(6-)(mnt)(1-)˙](4-)˙, (1) (mnt, maleonitrile dithiolate) which evolves H(2)S gas on treatment with acid under ambient conditions has been synthesized and structurally characterized. The Fe-S distances in 1 are in the range 2.246-2.383 Å, in stark contrast to that of the known n = -2 member of the series based on the [Fe(4)(μ(3)-S)(4)(S(2)C(2)R(2))(4)](n) unit (R = CF(3), Ph) with Fe-S bond lengths of 2.149-2.186 Å. The EPR of 1 displays very weak signals at g, 4.03 and 2.38 along with a strong S-based radical EPR signal at g, 2.003 associated with five structured components tentatively assigned to hyperfine interaction arising out of the naturally abundant (57)Fe with <A> = 88 G. The EPR profile resembles the reduced Fe-S cluster of CO inhibited Clostridium pasteurianum W5 hydrogenase or the Fe(4)S(4) centers of wild-type enzyme, IspH treated with HMBPP or IPP.

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Previous studies identified five nifH-like genes (nifH2 through nifH6) in Clostridium pasteurianum (strain W5), where the nifH1 gene encodes the nitrogenase iron protein. Transcripts of these nifH genes, with the exception of nifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by the nifH-like genes were not reported. The nifH2 and nifH6 genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region of nifH2 and nifH6 was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of the nif genes of single-colony isolates of C. pasteurianum suggest that the nifH6 designation should be eliminated. Here the size of mRNA from nifH2 and the detection of the NifH2 polypeptide in nitrogen-fixing cells of C. pasteurianum are reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that the nifH1 and nifH2 mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH of Azotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixing C. pasteurianum cells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixing C. pasteurianum cells.

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.

Ethane formation from acetylene as a potential test for vanadium nitrogenase in vivo

Azotobacter chroococcum and A. vinelandii, both obligately aerobic diazotrophic bacteria, have at least two genetically distinct systems for nitrogen fixation1,2. One is the well-characterized molybdenum nitrogenase, whereas in a second system (the vanadium nitrogenase) the conventional molybdoprotein is replaced by a vanadoprotein and a second iron protein replaces the one normally found with the molybdoprotein3–5. A third system may exist in A. vinelandii6'7. Reiterations of DNA encoding nitrogenase structural genes (nifHDK) are found in several genera of diazotrophs (see ref. 1). In the azotobacters there is evidence, based on physiological and biochemical properties of strains carrying deletions of nifHDK, that the reiterated genes are involved in the synthesis of alternative nitrogenases including the vanadium (V)-nitrogenase. The screening of other genera for the presence of a functional V-nitrogenase would be greatly facilitated by a specific test for it which could be applied in vivo. The reduction of acetylene to ethylene8,9 by the molybdenum (Mo)-nitrogenase has been widely used in ecological, genetic and physiological studies of biological nitrogen fixation. We demonstrate here that purified V-nitrogenase catalyses the reduction of acetylene not only to ethylene, but also to ethane, which the Mo-nitrogenase does not10. This property distinguishes between these two systems in A. chroococcum and A. vinelandii in vivo. Ethane formation also occurred in cultures of Clostridium pasteurianum W5, suggesting that V-nitrogenase is not restricted to the Azotobacteriaceae.

Purification and characterization of a molybdenum-pterin-binding protein (Mop) in Clostridium pasteurianum W5.

A large-scale fractionation scheme purified the major molybdenum(Mo)-binding protein (Mop) from crude extracts of Clostridium pasteurianum, with a 10 and 0.2% yield of Mo and protein, respectively. The apparent molecular weight of the purified molybdoprotein is 5,700, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein contains 0.7 mol of Mo per mol of protein with a molecular weight of 5,700. Mop, as isolated, has a peak absorbency at 293 nm. Denaturation and oxidation of the molybdoprotein released multiple pterin like fluorescent compounds. Mop appears to contain a pterin derivative and Mo, but phosphate analysis indicated that the pterin at the very least is not phosphorylated; phosphorylation is required for functional molybdenum cofactor. All treatments used to release the putative Mo-pterin species from Mop failed to yield a molybdopterin that had detectable molybdenum cofactor activity.

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.

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.

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.

Purification and properties of the H 2-oxidizing (uptake) hydrogenase of the N 2-fixing anaerobe Clostridium pasteurianum W5

Clostridium pasteurianum has two distinct hydrogenases, the bidirectional hydrogenase and the H 2-oxidizing (uptake) hydrogenase. The H 2-oxidizing hydrogenase has been purified (up to 970-fold) to a specific activity of 17,600 μmol H 2 oxidized/min·mg protein (5 mM methylene blue) or 3.5 μmol H 2 produced/min·mg protein (1 mM methyl viologen). The uptake hydrogenase has a M r of 53,000 (one polypeptide chain). Depending upon how protein was measured, the Fe and S = contents (gatom/mol) were 4.7 and 5.2 (by the dye-binding assay) or 7.2 and 8.0 (by the Lowry method). Both reduced and oxidized forms of the enzyme gave electron paramagnetic resonance signals. The activation energy for H 2-production and H 2-oxidation by the uptake hydrogenase was 59.1 and 31.2 kJ/mol, respectively. In the exponential phase of growth, the ratio of uptake hydrogenase/bidirectional hydrogenase in NH 3-grown cells was much lower than that in N 2-fixing cells.

Isolation of thiomolybdate compounds from the molybdenum-iron protein of clostridial nitrogenase.

Acid/base treatment of the molybdenum-iron protein of the nitrogenase from Clostridium pasteurianum 25 yields low-molecular-weight compounds of molybdenum, which can be separated from the protein by gel chromatography. Elementary analysis and spectral properties relate these compounds to thiomolybdate anions. It is proposed that in its native state nitrogenase contains a thio complex of molybdenum coupled to iron-sulfur clusters.

Isolation and properties of a unidirectional H 2-oxidizing hydrogenase from the strictly anaerobic N 2-fixing bacterium [formula omitted] W5

A unidirectional H 2-oxidizing hydrogenase (H 2ase-HO) has been isolated from the anaerobic N 2-fixer Clostridium pasteurianum W5. Extensively purified (> 200-fold or > 400–800 μmoles H 2 oxidized/min/mg protein) H 2ase-HO can use ferredoxin (Fd), methyl viologen (MV), benzyl viologen, methylene blue or dichlorophenolindophenol as the sole electron acceptor but does not produce H 2 from reduced Fd or MV. H 2ase-HO has a lower mol. wt., is less negatively charged and less O 2-sensitive than the classical bidirectional H 2ase from the same organism. The level of the H 2-oxidizing enzyme is higher in N 2-fixing cells than in NH 3-grown cells. H 2ase-HO occurs mainly outside the cell membrane and is inhibited by carbon monoxide.

Purification and properties of paramagnetic protein from clostridium pasteurianum W5

The purification to homogeneity of the non-heme iron protein, sometimes referred to as either “red protein’ or “paramagnetic protein’, from Clostridium pasteurianum W5 extracts is described and its physicochemical properties studied. This paramagnetic protein ( g  1.94) has a molecular weight of about 25 000 and contains two iron and two acid-labile sulfur atoms per mol of protein. Its midpoint potential at pH 7.5, as determined by electron paramagnetic resonance titration, is —300 mV. Optical circular dichroism and electron paramagnetic resonance spectra of the paramagnetic protein are similar to those of two iron-two acid-labile sulfur ferredoxins. The biochemical reduction of the purified protein was also studied.

On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5.

Hydrogenase, purified to an average specific activity of 328 mumol of H2 evolved/(min X mg of protein) from Clostridium pasteurianum W5, was found to have 4-5 Fe and 4-5 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 Fe per molecule. Displacement of the iron-sulfur cluster from hydrogenase by thiophenol in 80% hexamethyl phosphoramide:20% H2O yielded the Fe4S4 (thiophenyl)4 dianion according to absorption spectroscopy. Electron paramagnetic resonance spectroscopy at 12 K showed that the iron-sulfur cluster in the enzyme could be reduced by the H2 to a state (g-values of 2.098, 1.970, and 1.898) similar to that in reduced ferredoxin and could be oxidized by dichlorophenolindophenol or H+ to a state (g-values at 2.099, 2.041, and 2.001) similar to that in high potential iron-sulfur proteins. These oxidations and reductions appeared to occur within the turnover time of the enzyme. Deuterium failed to narrow the electron paramagnetic resonance signal in either state, but the competitive inhibitor carbon monoxide reversibly formed a compound with either state and substantially altered the electron paramagnetic resonance. 13CO produced a broadening of these signals, suggesting the formation of a direct CO complex with the iron-sulfur cluster. These data are consistent with a model of the active site of the enzyme in which a four-iron four-sulfur cluster is a component that can accept one or two electrons from and donate either one or two electrons to substrates, and in which the iron-sulfur cluster serves as the site of binding of gaseous ligands.

Purification and properties of hydrogenase from Clostridium pasteurianum W5

Hydrogenase from Clostridium pasteurianum W5 prepared by a previously reported method was found to be heterogeneous. A new procedure has been developed which achieved a 320-fold purification with an 11% recovery of the enzyme activity. This represents a 6.6-fold increase in specific activity over the previously reported value. Such preparations showed a maximal activity ( V) of about 4000 μmoles H 2 evolved/min/mg protein when assayed in the presence of ferredoxin and 1 mM methyl viologen. Hydrogenase so prepared has a molecular weight of 60 500 and did not dissociate into subunits when treated with sodium dodecylsulfate and 2-mercaptoethanol. Hydrogenase has 12 half-cystine residues per molecule and a preponderance of acidic amino acids. This hydrogenase does not contain molybdenum or copper but has about 12 iron atoms and 12 acid-labile sulfur groups per molecule. All of the iron atoms are resistant to anaerobic treatment with o- phenanthroline . Methyl viologen, benzyl viologen, methylene blue and ferredoxin can each serve as the sole electron carrier for the purified enzyme.

Evidence for the Existence of a Fully Reduced State of Molybdoferredoxin during the Functioning of Nitrogenase, and the Order of Electron Transfer from Reduced Ferredoxin

Abstract Molybdoferredoxin from Clostridium pasteurianum W5 was studied under conditions where the nitrogenase system was turning over and molybdoferredoxin was known to be ∼90% in the EPR-silent state. Optical spectroscopy showed that the EPR-silent molybdoferredoxin species thus formed was not the same as the EPR-silent species formed by dye oxidation. The steady state (EPR-silent) molybdoferredoxin and the EPR-positive form of molybdoferredoxin isolated from cells in the presence of dithionite were oxidized with a 1.3-fold excess thionin and the numbers of electrons transferred to thionin were calculated. It was found that the EPR-silent molybdoferredoxin species formed by incubation with azoferredoxin, MgATP, and dithionite held 4 more transferable electrons per tetramer (2 more per dimer) than EPR-positive molybdoferredoxin. Oxidized azoferredoxin could be reduced by reduced ferredoxin, whereas oxidized molybdoferredoxin could not be reduced by reduced ferredoxin unless both azoferredoxin and MgATP were present. A reduction scheme for N2 fixation is proposed.

Oxidation reduction properties of nitrogenase from [formula omitted] W5

Dithionite reduced azoferredoxin and molybdoferredoxin from Clostridium pasteurianum W5 were oxidatively titrated with various electron acceptors. The AzoFd gave up 0.87 electrons per AzoFd monomer (27,500 mol. wt.). The oxidation reduction potential of AzoFd, determined by equilibrium with redox dyes, was −0.240 V. Dithionite reduced MoFd gave up 3.6 electrons per MoFd tetramer (220,000 mol. wt.). The oxidation reduction potential for MoFd was −0.070 V. Because the potential of this MoFd half cell is so positive, the electrons removed during this oxidation may not be those that reduce dinitrogen.

Electron paramagnetic resonance of nitrogenase and nitrogenase components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP

Electron paramagnetic resonance of nitrogenase and nitrogenase components from <i>Clostridium pasteurianum W5</i> and <i>Azotobacter vinelandii OP</i>

On the structure and function of nitrogenase from [formula omitted] W5

Molybdoferredoxin from Clostridium pasteurianum W5 was fractionated into MoFd with two atoms of molybdenum per 220,000 daltons and a specific activity of 2.6 μmoles C 2H 2 reduced/min/mg protein and into a catalytically inactive species with an identical protein moiety but an incomplete active centre. Native MoFd is a tetramer composed of two 50,000 and two 60,000 dalton subunits. At low protein concentrations the tetramer is in equilibrium with a dimer. Under low ionic strength and at low pH further dissociation into monomers occurs. MoFd and azoferredoxin have distinct electron paramagnetic resonance spectra. The EPR spectrum of AzoFd and that of the combination of the two nitrogenase components undergoes characteristic changes upon addition of MgATP 2−.

Molecular weight and subunit structure of molybdoferredoxin from Clostridium pasteurianum W5

1. 1. Molybdoferredoxin, a component of the nitrogenase system of Clostridium pasteurianum W5, has a molecular weight of between 160 000 to 190 000 as estimated by gel filtration. 2. 2. Sodium dodecyl sulfate-treated molybdoferredoxin gives two bands on electrophoresis, one of which has a molecular weight of 59 500 ± 1940 and the other a molecular weight of 50 700 ± 1950. Microdensitometer tracings of the two bands and estimation of their areas suggest that there is a ratio of two subunits of the heavier protein to one subunit of the lighter protein. This argues that the molecular weight of molybdoferredoxin is about 170 000 and it contains two 59 500 and one 50 700 subunits.

Mutant of Clostridium pasteurianum that does not fix nitrogen.

A stable, non-nitrogen-fixing mutant of Clostridium pasteurianum W5 was isolated. The mutant has the same growth rate as the wild type on ammonia nitrogen, but is not able to grow on N(2). Cell-free extracts of the derepressed mutant are unable to fix nitrogen, or reduce the alternate substrates of nitrogenase such as cyanide, azide, or acetylene. Recombination with wild-type components from a diethylaminoethyl cellulose column indicates that the mutant contains component II, but is lacking component I activity.