Klaus Schneider

Bielefeld University, Bielefeld, North Rhine-Westphalia, Germany

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Publications (33)114.76 Total impact

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    ABSTRACT: The molybdenum storage protein (MoSto) can store more than 100 Mo or W atoms as discrete polyoxometalate (POM) clusters. Here, we describe the three POM cluster sites along the threefold axis of the protein complex based on four X-ray structures with slightly different polyoxomolybdate compositions between 1.35 and 2 Å resolution. In contrast to the Moα-out binding site occupied by an Mo3 cluster, the Moα-in and Moβ binding sites contain rather weak and non-uniform electron density for the Mo atoms (but clearly identifiable by anomalous data), suggesting the presence of POM cluster ensembles and/or degradation products of larger aggregates. The “Moα-in cluster ensemble” was interpreted as an antiprism-like Mo6 species superimposed with an Mo7 pyramide and the “Moβ cluster ensemble” as an Mo13 cluster (present mostly in a degraded form) composed of a pyramidal Mo7 and a Mo3 building block linked by three spatially separated MoOx units. Inside the ball-shaped Mo13 cluster sits an occluded central atom, perhaps a metal ion. POM cluster formation at the Moα-in and Moβ sites appears to be driven by filtering out and binding/protecting self-assembled transient species complementary to the protein template.
    Journal of Inorganic Biochemistry 09/2014; 138. DOI:10.1016/j.jinorgbio.2014.05.009 · 3.27 Impact Factor
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    ABSTRACT: Some N(2)-fixing bacteria prolong the functionality of nitrogenase in molybdenum starvation by a special Mo storage protein (MoSto) that can store more than 100 Mo atoms. The presented 1.6 Å X-ray structure of MoSto from Azotobacter vinelandii reveals various discrete polyoxomolybdate clusters, three covalently and three noncovalently bound Mo(8), three Mo(5-7), and one Mo(3) clusters, and several low occupied, so far undefinable clusters, which are embedded in specific pockets inside a locked cage-shaped (αβ)(3) protein complex. The structurally identical Mo(8) clusters (three layers of two, four, and two MoO(n) octahedra) are distinguishable from the [Mo(8)O(26)](4-) cluster formed in acidic solutions by two displaced MoO(n) octahedra implicating three kinetically labile terminal ligands. Stabilization in the covalent Mo(8) cluster is achieved by Mo bonding to Hisα156-N(ε2) and Gluα129-O(ε1). The absence of covalent protein interactions in the noncovalent Mo(8) cluster is compensated by a more extended hydrogen-bond network involving three pronounced histidines. One displaced MoO(n) octahedron might serve as nucleation site for an inhomogeneous Mo(5-7) cluster largely surrounded by bulk solvent. In the Mo(3) cluster located on the 3-fold axis, the three accurately positioned His140-N(ε2) atoms of the α subunits coordinate to the Mo atoms. The formed polyoxomolybdate clusters of MoSto, not detectable in bulk solvent, are the result of an interplay between self- and protein-driven assembly processes that unite inorganic supramolecular and protein chemistry in a host-guest system. Template, nucleation/protection, and catalyst functions of the polypeptide as well as perspectives for designing new clusters are discussed.
    Journal of the American Chemical Society 05/2012; 134(23):9768-74. DOI:10.1021/ja303084n · 11.44 Impact Factor
  • Acta Crystallographica Section A Foundations of Crystallography 08/2011; 67(a1):C217-C217. DOI:10.1107/S0108767311094621 · 2.07 Impact Factor
  • Klaus Schneider, Achim Müller
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    ABSTRACT: „Pflanzen-Bakterien-Symbiosen”, „N2Fixierung und Ökologie” und „N2-Fixierung in Landwirtschaft und Industrie” waren die Hauptschwerpunkte beim „8th International Congress on Nitrogen Fixation”, der vom 20. bis 26. Mai in Knoxville, Tennessee/USA, stattfand. Die Themen entsprechen der pflanzenphysiologisch - landwirtschaftlichen Ausrichtung der Arbeitsgruppe von Prof. P. M. Gresshoff (Institut of Agriculture and Center of Legume Research, Knoxville). Die aus der Sicht der Chemie und Biochemie besonders interessanten Themenbereiche betrafen zum einen Struktur und Lokalisation der Metall-Cluster in der Nitrogenase und zum anderen die Charakterisierung alternativer, d. h. Mo-unabhängiger Nitrogenasen.
    Nachrichten aus der Chemie 09/2010; 38(9):1076-1077. DOI:10.1002/nadc.19900380910 · 0.20 Impact Factor
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    ABSTRACT: The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12 degrees C revealed that three different steps can be distinguished for the Mo-release process. A proportion of approximately 15% at pH 6.8-7.0, an additional 25% at pH 7.2-7.5 and ca. 50% (up to 90% in total) at pH 7.6-7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30 degrees C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play-in addition to the pH value-an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110-115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12 degrees C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but could also be constructed in vitro by a metalate-ion exchange procedure by using the isolated MoSto protein. The high W content of the isolated cell-made WSto (approximately 110 atoms/protein molecule) and the relatively low amount of tungstate that was released from the protein under optimal "release conditions", demonstrates that the W-oxide-based clusters are more stable inside the protein cavity than the Mo-oxide analogues, as expected from the corresponding findings in polyoxometalate chemistry. The optimized isolation of the W-loaded protein form allowed us to get single crystals, and to determine the crystal X-ray structure. This proved that the protein contains remarkably different types of polyoxotungstates, the formation of which is templated in an unprecedented process by the different protein pockets. (Angew. Chem. Int. Ed. 2007, 46, 2408-2413).
    ChemBioChem 03/2008; 9(4):595-602. DOI:10.1002/cbic.200700446 · 3.06 Impact Factor
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    ABSTRACT: We thank Prof. H. Michel for continuous support, Dr. H. Bögge for helpful discussions and the staff of the X6SA and X10SA beamlines (in particular Dr. E. Pohl and Dr. C. Schulze-Briese) at the Swiss-Light-Source, Villigen for assistance during data collection. A.M. gratefully acknowledges the very generous support by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
    Angewandte Chemie International Edition 03/2007; 46(14):2408-13. DOI:10.1002/anie.200604858 · 11.34 Impact Factor
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    ABSTRACT: The membrane-bound hydrogenase from Paracoccus dentitrificans was purified 68-fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 μmol H2 min−1 (mg protein)−1 (methylene blue reduction). Purification involved solubilzation by Triton X-114, phase separation, chromatography on DEAE-Sephacel, ammonium-sulfate precipitation and chromatography on Procion-red HE-3B-Sepharose. Gel electrophoresis under denatruing conditions revealed two non-identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane-bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half-life for inactivation under air at 200C was 8 h.The Praracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6-dichloriondophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V= 161.9 U/mg; Km= 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively).The native hydrogenase from P. denitrificans cross-reacted with purified antibodies raised aginst the membrane-bound hydrogense from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.
    03/2005; 179(1):101 - 108. DOI:10.1111/j.1432-1033.1989.tb14526.x
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    ABSTRACT: Azotobacter vinelandii is a diazotrophic bacterium characterized by the outstanding capability of storing Mo in a special storage protein, which guarantees Mo-dependent nitrogen fixation even under growth conditions of extreme Mo starvation. The Mo storage protein is constitutively synthesized with respect to the nitrogen source and is regulated by molybdenum at an extremely low concentration level (0-50 nM). This protein was isolated as an alpha4beta4 octamer with a total molecular mass of about 240 kg mol(-1) and its shape was determined by small-angle X-ray scattering. The genes of the alpha and beta subunits were unequivocally identified; the amino acid sequences thereby determined reveal that the Mo storage protein is not related to any other known molybdoprotein. Each protein molecule can store at least 90 Mo atoms. Extended X-ray absorption fine-structure spectroscopy identified a metal-oxygen cluster bound to the Mo storage protein. The binding of Mo (biosynthesis and incorporation of the cluster) is dependent on adenosine triphosphate (ATP); Mo release is ATP-independent but pH-regulated, occurring only above pH 7.1. This Mo storage protein is the only known noniron metal storage system in the biosphere containing a metal-oxygen cluster.
    ChemBioChem 02/2005; 6(2):405-13. DOI:10.1002/cbic.200400263 · 3.06 Impact Factor
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    ABSTRACT: Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.
    Journal of Bacteriology 02/2005; 187(1):92-8. DOI:10.1128/JB.187.1.92-98.2005 · 2.69 Impact Factor
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    ABSTRACT: In the phototrophic non-sulfur bacterium Rhodobacter capsulatus, the biosynthesis of the conventional Mo-nitrogenase is strictly Mo-regulated. Significant amounts of both dinitrogenase and dinitrogenase reductase were only formed when the growth medium was supplemented with molybdate (1 microM). During cell growth under Mo-deficient conditions, tungstate, at high concentrations (1 mM), was capable of partially (approximately 25%) substituting for molybdate in the induction of nitrogenase synthesis. On the basis of such conditions, a tungsten-substituted nitrogenase was isolated from R. capsulatus with the aid of anfA (Fe-only nitrogenase defective) mutant cells and partially purified by Q-sepharose chromatography. Metal analyses revealed the protein to contain an average of 1 W-, 16 Fe-, and less than 0.01 Mo atoms per alpha(2)beta(2)-tetramer. The tungsten-substituted (WFe) protein was inactive in reducing N(2) and marginally active in acetylene reduction, but it was found to show considerable activity with respect to the generation of H(2) from protons. The EPR spectrum of the WFe protein, recorded at 4 K, exhibited three distinct signals: (i) an S = 3/2 signal, which dominates the low-field region of the spectrum (g = 4.19, 3.93) and is indicative of a tungsten-substituted cofactor (termed FeWco), (ii) a marginal S = 3/2 signal (g = 4.29, 3.67) that can be attributed to residual amounts of FeMoco present in the protein, and (iii) a broad S = 1/2 signal (g = 2.09, 1.95, 1.86) arising from at least two paramagnetic species. Redox titrational analysis of the WFe protein revealed the midpoint potential of the FeWco (E(m) < -200 mV) to be shifted to distinctly lower potentials as compared to that of the FeMoco (E(m) approximately -50 mV) present in the native enzyme. The P clusters of both the WFe and the MoFe protein appear indistinguishable with respect to their midpoint potentials. EPR spectra recorded with the WFe protein under turnover conditions exhibited a 20% decrease in the intensity of the FeWco signal, indicating that the cofactor can be enzymatically reduced only to a small extent. The data presented in the current study demonstrate the pivotal role of molybdenum in optimal N(2) fixation and provides direct evidence that the inability of a tungsten-substituted nitrogenase to reduce N(2) is due to the difficulty to effectively reduce the FeW cofactor beyond its semi-reduced state.
    Biochemistry 05/2003; 42(13):3846-57. DOI:10.1021/bi0270790 · 3.19 Impact Factor
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    ABSTRACT: The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of −195 mV (appearance) and −30 mV (disappearance), (2) an axial signal (g|| = 2.00, g⊥ = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the ‘low-potential’ rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the ‘high-potential’ rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: −80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this ‘turnover signal’ nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.
    03/2002; 269(6):1650 - 1661. DOI:10.1046/j.1432-1327.2002.02804.x
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    ABSTRACT: The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of -195 mV (appearance) and -30 mV (disappearance), (2) an axial signal (g(parallel) = 2.00, g perpendicular = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the 'low-potential' rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the 'high-potential' rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exhibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: -80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this 'turnover signal' nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.
    European Journal of Biochemistry 03/2002; 269(6):1650-61. · 3.58 Impact Factor
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    ABSTRACT: In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.
    European Journal of Biochemistry 05/2001; 268(7):1940-52. DOI:10.1046/j.1432-1327.2001.02063.x · 3.58 Impact Factor
  • Angewandte Chemie International Edition 09/1997; 36(16):1747-1750. DOI:10.1002/anie.199717471 · 11.34 Impact Factor
  • Angewandte Chemie 08/1997; 109(16):1812-1816. DOI:10.1002/ange.19971091620
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    ABSTRACT: The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (delta nifHDK, delta modABCD strain) and purified in a one-day procedure that included only one column-chromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, Rc1Fe), which was more than 95% pure, and an approximately 80% pure component 2 (Fe protein, Rc2Fe). The highest specific activities, which were achieved at an Rc2Fe/Rc1Fe molar ratio of 40:1, were 260 (C2H4 from C2H2), 350 (NH3 formation), and 2400 (H2 evolution) nmol product formed x min(-1) x mg protein(-1). The purified FeFe protein contained 26 +/- 4 Fe atoms; it did not contain Mo, V, or any other heterometal atom. The most significant catalytic property of the iron-only nitrogenase is its high H2-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N2 reduction, the activity ratios (mol N2 reduced/mol H2 produced) obtained were 1:1 (molybdenum nitrogenase) and 1:7.5 (iron nitrogenase). The Rc1Fe protein has only a very low affinity for C2H2. The Km value determined (12.5 kPa), was about ninefold higher than the Km for Rc1Mo (1.4 kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5. In complementation experiments, component 1 proteins coupled very poorly with the 'wrong' component 2. Rc1Fe, if complemented with Rc2Mo, showed only 10-15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rc1Fe and Rc1Mo are immunologically not related. The most active Rc1Fe samples appeared to be EPR-silent in the Na2S2O4-reduced state. However, on partial oxidation with K3[Fe(CN)6] or thionine several signals occurred. The most significant signal appears to be the one at g = 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between -100 mV and +150 mV. Another novel EPR signal (g = 1.96, 1.92, 1.77) occurred on further reduction of Rc1Fe by using turnover conditions in the presence of a substrate (N2, C2H2, H+).
    European Journal of Biochemistry 04/1997; 244(3):789-800. · 3.58 Impact Factor
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    ABSTRACT: The effect of Cd2+, Pb2+, Hg2+ and Cu2+ on the aggregation behaviour of monoclonal rat-IgG1-anti-mouse antibodies (kappa-light chain specific) and their antibody-antigen complexes with monoclonal mouse-IgG1 is reported. Investigations were done using the dynamic light scattering method. Cd2+ ions affected the hydrodynamic properties of the antibodies and the immune complex formation very little. More than 4 Cu2+ ions per antibody molecule led to large insoluble aggregates. Pb2+ ions also interacted with antibodies and immune complexes. Instead of "monomeric' antibodies (Ab) or immune complexes (Ab1Ag1), large soluble aggregates were detectable in the solution. Hg2+ ions induced complex formation with 3-4 antibodies per aggregate. Possible kinds of interaction are discussed. Additionally, we tested the antigen binding activity of metal-treated antibodies in ELISA-tests. The Sandwich ELISA technique was used to investigate the serological activity of the metal-treated antibodies, i.e., the reaction with the specific antigen. For these experiments we used the same monoclonal antibodies, mouse-IgG1 and rat-IgG1-anti-mouse. The influence of the above mentioned heavy metal ions was investigated up to a 10-fold molar excess over the antibody concentration. Even at these "unphysiological' high metal ion concentrations an inhibition of the antibody-antigen binding activity was not detectable.
    Biochimica et Biophysica Acta 03/1997; 1334(1):98-108. DOI:10.1016/S0304-4165(96)00078-5 · 4.66 Impact Factor
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    ABSTRACT: Experimental results for the nitrogenase MoFe protein from Azotobacter vinelandii obtained by dynamic light scattering (DLS) are presented. The translational diffusion coefficient was determined to D = (4.0 +/- 0.2) x 10(-7) cm2/s. Complementary, we have performed hydrodynamic model calculations based on the X-ray crystallographic data of the MoFe protein. The calculated transport coefficient suggests that the size and shape of the protein in solution is consistent with that in the crystal structure.
    Biochimica et Biophysica Acta 03/1997; 1337(2):311-8. DOI:10.1016/S0167-4838(96)00179-3 · 4.66 Impact Factor
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    ABSTRACT: The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (ΔnifHDK, μModABCD strain) and purified in a one-day procedure that included only one column-chromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, Rc1Fe), which was more than 95% pure, and an approximately 80 % pure component 2 (Fe protein, Rc2Fe). The highest specific activities, which were achieved at an Rc2Fe/Rc1Fe molar ratio of 40:1, were 260 (C2H4 from C2H2), 350 (NH3 formation), and 2400 (H2 evolution) nmol product formed min−1 mg protein−1. The purified FeFe protein contained 26 ± 4 Fe atoms; it did not contain Mo, V, or any other heterometal atom.The most significant catalytic property of the iron-only nitrogenase is its high H2-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N2 reduction, the activity ratios (mol N2 reduced/mol H2 produced) obtained were 1:1 (molybdenum nitrogenase) and 1:7.5 (iron nitrogenase). The Rc1Fe protein has only a very low affinity for C2H2. The Km value determined (12.5 kPa), was about ninefold higher than the Km for Rc1Mo (1.4kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5.In complementation experiments, component 1 proteins coupled very poorly with the ‘wrong’ component 2. Rc1Fe, if complemented with Rc2Mo, showed only 10–15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rc1Fe and Rc1Mo are immunologically not related.The most active Rc1Fe samples appeared to be EPR-silent in the Na2S2O4-reduced state. However, on partial oxidation with K3[Fe(CN)6] or thionine several signals occurred. The most significant signal appears to be the one at g= 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between -100 mV and +150 mV. Another novel EPR signal (g= 1.96, 1.92, 1.77) occurred on further reduction of Rc1Fe by using turnover conditions in the presence of a substrate (N2, C2H2, H+).
    02/1997; 244(3):789 - 800. DOI:10.1111/j.1432-1033.1997.t01-1-00789.x
  • E. Krahn, K. Schneider, A. Müller
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    ABSTRACT:  Cell suspensions of uptake-hydrogenase-deficient (hup -) mutants of a wild-type (B10S) and a nifHDK deletion strain of Rhodobacter capsulatus were used comparatively to characterize the conventional, Mo-containing and the alternative, “iron-only” nitrogenase of this organism by determining the H2 production and acetylene reduction activities under argon and dinitrogen atmospheres. A comparison with the corresponding hup + strains revealed that the hup - mutation did not affect the nitrogenase activity and specificity within the acetylene-reduction assay, but caused a significant increase in H2 production, which was more prominent in the case of the ΔnifHDK strain. The ΔnifHDK hup - cells, grown in Mo-depleted medium and, thus, expressing the alternative nitrogenase system, were more than ten times less active in the acetylene-reduction assay but exhibited H2 production rates equivalent to about 60% of the rates obtained with B10S hup - cells after growth in a medium containing 10 μM MoO- 4. When these conditions were applied, the B10S strain only expressed the Mo nitrogenase. Under an argon atmosphere containing about 5.5% (v/v) acetylene and under a dinitrogen atmosphere, ΔnifHDK hup - cells produced H2 at even higher rates than B10S hup - cells. The implications of our findings on a possible biotechnological H2 production and on the mechanism of nitrogenase catalysis are considered.
    Applied Microbiology and Biotechnology 09/1996; 46(3):285-290. DOI:10.1007/s002530050818 · 3.81 Impact Factor