Oxidation reduction properties of nitrogenase from Clostridium pasteurianum W5

Dithionite reduced azoferredoxin and molybdoferredoxin from $\text{Clostridium pasteurianum}$ 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 $\text{this}$ MoFd half cell is so positive, the electrons removed during this oxidation may not be those that reduce dinitrogen.     

Purification and characterization of an inducible dissimilatory type sulfite reductase from Clostridium pasteurianum

An inducible sulfite reductase was purified from Clostridium pasteurianum. The pH optimum of the enzyme is 7.5 in phosphate buffer. The molecular weight of the reductase was determined to be 83,600 from sodium dodecyl sulfate gel electrophoresis with a proposed molecular structure: ₂₂. Its absorption spectrum showed a maximum at 275 nm, a broad shoulder at 370 nm and a very small absorption maximum at 585 nm. No siroheme chromophore was isolated from this reductase. The enzyme could reduced the following substrates in preferential order: NH₂OH> SeO ₃ ^2- >NO ₂ ^2- at rates 50% or less of its preferred substrate SO ₃ ^2- . The proposed dissimilatory intermediates, S₃O ₆ ^2- or S₂O ₃ ^2- , were not utilized by this reductase while KCN inhibited its activity. Varying the substrate concentration [SO ₃ ^2- ] from 1 to 2.5 mol affected the stoichiometry of the enzyme reaction by alteration of the ratio of H₂ uptake to S^2- formed from 2.5:1 to 3.1:1. The inducible sulfite reductase was found to be linked to ferredoxin which could be completely replaced by methyl viologen or partially by benzyl viologen. Some of the above-mentioned enzyme properties and physiological considerations indicated that it was a dissimilatory type sulfite reductase.Abbreviations SDS sodium dodecyl sulfate - BSA bovine serum albumin - LDH Lactate dehydrogenase     

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 M?ssbauer 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.     

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

Previous M?ssbauer 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 M?ssbauer 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.     

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.     

Purification and characterization of placental protein 5

This report describes the purification of placental protein 5, PP5, from the human placenta by two affinity chromatography steps, the first with Heparin-Sepharose and the second with Sepharose-linked monoclonal anti-PP5 antibody. The final purification is achieved by reversed-phase high performance liquid chromatography. In SDS-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, PP5 purified in this study migrates as one major band at 36 kD. The previously purified PP5 is more heterogeneous: under nonreducing conditions it migrates at 30 kD and, after reduction, it gives three bands at 16.8 kD, 18.3 kD, and 19.0 kD. In Western blot analysis, both purified proteins react with polyclonal and monoclonal anti-PP5 antibodies. Three N-terminal amino acid sequences are obtained for the previously purified PP5, whereas the N-terminal of PP5 purified in this study is blocked. These results suggest that PP5 previously purified in the absence of protease inhibitors, does not represent the native form of PP5. Computer comparison of the obtained amino acid sequences revealed no significant homology to known protein sequences.     

Purification and characterization of an endoglucanase (1,4-beta-D-glucan glucanohydrolase) from Clostridium thermocellum.

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) was purified from Clostridium thermocellum by procedures that included centrifugation, ultrafiltration, selective precipitation, ion-exchange Sephadex chromatography and preparative gel electrophoresis. The 22-fold-purified enzyme behaved as a homogeneous protein under non-denaturing conditions. The enzyme represented a significant component (greater than 25%) of total extracellular endoglucanase activity, but was purified in low yield by the procedures employed. The native molecular weight of the endoglucanase was determined by ultracentrifugational analysis, amino acid composition and polyacrylamide-gel electrophoresis, and varied between 83000 and 94000. The enzyme contained 11.2% carbohydrate and was isoelectric at pH 6.72. The pH and temperature optima of the endoglucanase were 5.2 and 62 degrees C respectively. The enzyme lacked cysteine and was low in sulphur-containing amino acids. The purified endoglucanase displayed: high activity towards carboxymethylcellulose, celloheptaose, cellohexaose and cellopentaose; low activity towards Avicel microcrystalline cellulose and cellotetraose; no detectable activity towards cellotriose or cellobiose; increased activity towards cello-oligosaccharides with increasing degree of polymerization. The internal glycosidic bonds of cello-oligosaccharides were cleaved by the enzyme in preference to external linkages. The apparent Michaelis constant ([S]0.5V) and Vmax. for cellopentaose and cellohexaose hydrolysis were 2.30 mM and 39.3 mumol/min per mg of protein, and 0.56 mM and 58.7 mumol/min per mg of protein, respectively.     

Production of Clostridium pasteurianum in a Defined Medium

A method for the growth of Clostridium pasteurianum in a 140-liter (total capacity) stainless-steel vessel is described. By preventing the pH value from falling below 5.6, the growth of cultures was prolonged. Larger amounts of the carbon source (sucrose) and the nitrogen source (ammonium ion) were supplied and consumed, and cell yields of up to 5.56 g (dry weight) per liter were obtained. The highest cell yield previously reported was 1.7 g (dry weight) per liter obtained under nitrogen-fixing conditions in 500-ml cultures. The ferredoxin content of the cells was comparable with that obtained by earlier workers.     

Purification and Properties of the Constituents of the Nitrogenase Complex from Clostridium pasteurianum

A new procedure for a rapid and extensive purification of the FeMo protein and the Fe protein of the nitrogenase complex from Clostridium pasteurianum is described. Specific activities of 345 and 460 nmoles of N(2) reduced per mg of protein per min for the FeMo protein and for the Fe protein, respectively, have been obtained. Preparations of the FeMo protein contained 0.96 atom of molybdenum and 15 atoms of iron per molecule, whereas those of the Fe protein contained 2.86 atoms of iron per molecule. Experiments suggest that a definite association of two Fe proteins and one FeMo protein is functional in the active enzyme complex. No individual role could be ascribed to either of the two proteins, but the fact that hydrogenase inhibits N(2) fixation but not the reductant-dependent adenosine triphosphate hydrolysis supports the idea that there are two distinct sites on nitrogenase, one concerned with N(2) activation and the other with activated electron transport.