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At least 19 recordsLinked to original sources
PubMed1978

Nitrogenase and nitrogenase reductase associate and dissociate with each catalytic cycle.

Nitrogenase and nitrogenase reductase dissociate after each electron is transferred between them, as shown by the occurrence of a lag phase approximately as long as the average turnover time of nitrogenase before hydrogen evolution occurs. Because nitrogenase was present in the reaction mixture in large excess over nitrogenase reductase, the electrons donated by nitrogenase reductase must have been distributed randomly over all of the nitrogenase present. This is accomplished by nitrogenase reductase molecules associating randomly with nitrogenase molecules for each cycle of electrons transferred. The fact that ATP is hydrolyzed without a lag indicates both that electron transfer occurs during the lag and the ATP hydrolysis is coupled to electron transfer from nitrogenase reductase to nitrogenase and not to substrate reduction. The observations support the suggestion that it now is desirable to alter nomenclature to designate the MoFe protein as nitrogenase and the Fe protein as nitrogenase reductase.

Adenosine Triphosphate↗
PubMed1996

Pre-steady-state kinetics of nitrogenase from Azotobacter vinelandii. Evidence for an ATP-induced conformational change of the nitrogenase complex as part of the reaction mechanism.

The pre-steady-state electron transfer reactions of nitrogenase from Azotobacter vinelandii have been studied by stopped-flow spectrophotometry. With reduced nitrogenase proteins after the initial absorbance increase at 430 nm (which is associated with electron transfer from the Fe protein to the MoFe protein and is complete in 50 ms) the absorbance decreases, which, dependent on the ratio [Av2]/[Av1], is followed by an increase of the absorbance. The mixing of reductant-free nitrogenase proteins with MgATP leads after 20 ms to a decrease of the absorbance, which could be fitted (from 0. 05 to 1 s) with a single exponential decay with a rate constant kobs = 6.6 +/- 0.8 s-1. This reaction of nitrogenase was measured at different wavelengths. The data indicate the formation of a species with a blue shift of the absorbance of metal-sulfur clusters of nitrogenase from 430 to 360 nm. The absorbance decrease at 430 nm observed (after 50 ms) in the case of the reduced nitrogenase proteins could only be simulated well if, after the initial electron transfer from the Fe protein to the MoFe protein and before dissociation of the nitrogenase complex, an additional reaction was assumed. The rate constant of this reaction was of the same order as the rate constant of the MgATP-dependent pre-steady-state proton production by nitrogenase from A. vinelandii: kobs = 14 +/- 4 s-1 with reduced nitrogenase proteins and kobs = 6 +/- 2 s-1 with dithionite-free nitrogenase proteins (Duyvis, M. G., Wassink, H., and Haaker, H. (1994) Eur. J. Biochem. 225, 881-890). It is proposed that in the presence and absence of reductant, the observed absorbance decrease at 430 nm of nitrogenase is caused by a change of the conformation of the nitrogenase complex, as a consequence of hydrolysis of MgATP.

Adenosine Triphosphate↗
PubMed1993

Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor.

Nitrogenase-3 of Azotobacter vinelandii is synthesized under conditions of molybdenum and vanadium deficiency. The minimal metal requirement for its synthesis, and its metal content, indicated that the only transition metal in nitrogenase-3 was iron [Chisnell, Premakumar and Bishop (1988) J. Bacteriol. 170, 27-33; Pau, Mitchenall and Robson (1989) J. Bacteriol. 171, 124-129]. A new species of nitrogenase-3 has been purified from a strain of A. vinelandii (RP306) lacking structural genes for the Mo- and V-nitrogenases and containing a mutation which enables nitrogenase-3 to be synthesized in the presence of molybdenum. SDS/PAGE showed that component 1 contained a 15 kDa polypeptide which N-terminal amino acid sequence determination showed to be encoded by anfG. This confirms that nitrogenase-3, like V-nitrogenase, comprises three subunits. Preparations of the nitrogenase-3 from strain RP306 contained 24 Fe atoms and 1 Mo atom per molecule. Characterization of the cofactor centre of the enzyme by e.p.r. spectroscopy and an enzymic cofactor assay, together with stimulation of the growth of strain RP306 by Mo, showed that nitrogenase-3 can incorporate the Mo-nitrogenase cofactor (FeMoco) to form a functional enzyme. The specific activities (nmol of product produced/min per mg of protein) determined from activity titration curves were: under N2, NH3 formation 110, with concomitant H2 evolution of 220; under argon, H2 evolution 350; under 10% acetylene (C2H2) in argon, ethylene (C2H4) 58, ethane (C2H6) 26, and concomitant H2 evolution 226. The rate of formation of C2H6 was non-linear, and the C2H6/C2H4 ratio strongly dependent on the ratio of nitrogenase components.

Amino Acids↗
PubMed1988

Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase.

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.

Azotobacter↗
PubMed1995

The molybdenum nitrogenase from wild-type Xanthobacter autotrophicus exhibits properties reminiscent of alternative nitrogenases.

In the presence of molybdate (1 microM) 2-3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen-fixing hydrogen-oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm). The maximal specific activity obtained was 145 nmol ethylene reduced . min-1 . mg protein-1 (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate-starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X. autotrophicus, a Mo-independent nitrogenase does not exist. However, the molybdate-containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases. The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non-saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH-dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S = 3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.

Catalysis↗
PubMed1998

Effect of some physiological factors on nitrogenase activity and nitrogenase mediated hydrogen evolution by mixed microbial culture.

Fermentative H2 evolution, nitrogenase activity (acetylene reduction) and nitrogenase mediated H2 evolution was studied in free cells of mixed microbial population of H2 producers. At 3% glucose level, the cells produced 8.35 l H2/mol glucose utilized. The role of nitrogenase system in H2 generation was evident by derepressed nitrogenase activity (0.46 nmoles C2H4 produced/mg protein/h) under defined in vitro conditions. For maximum expression of the activity, the cells required preactivation under anaerobic conditions by incubating at 40 degrees C for 20-24 h with 0.2% glucose in the culture medium. At an O2 level of more than 0.25%, the acetylene reduction activity decreased significantly and could not be detected at a level of 20%. Nitrogenase activity development was higher at acetylene: inoculum ratio between 4.2-6.25. H2 evolution was lower when the mixed cells were incubated under an atmosphere of 10% C2H2 and 5% CO gas. This decrease in H2 evolution was also evident at 2.5-6.5 mM NaNO3 and KNO3 concentrations in the liquid culture medium thus establishing more than 50% H2 evolution through nitrogenase.

Acetylene↗
PubMed1993

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus.

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf- mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf- Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.

Amino Acid Sequence↗
PubMed1997

Comparative biochemical characterization of the iron-only nitrogenase and the molybdenum nitrogenase from Rhodobacter capsulatus.

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+).

Bacterial Proteins↗
PubMedDate not supplied

A model of nitrogenase active-centre and mechanism of nitrogenase catalysis.

Based upon the known reactions of nitrogenase and the principles of coordination catalysis, a model of nitrogenase active-centre is proposed. An octa-atomic cluster, Fe2S2-Mo2O2, of pseudo-cubane-type structure is supposed to form a coupled twin of trinuclear (2Mo-1Fe) active-centre, which also catalyzes the reduction of H+ to H2. With this model, mechanisms of all the known nitrogenase-catalyzed reactions are explained, together with the non-inhibition of the hydrogen-evolution reaction by CO, and the mixed character of inhibition of other nitrogenase-catalyzed reactions. Electron transport by 2-stepped ATP-driving with some electron back-flow is shown to give a reasonable explanation for the observed ATP/2e- ratio and for the reductant-independent ATP hydrolysis catalyzed by nitrogenase. The close analogy between electron transport by 2-stepped ATP-driving in nitrogenase catalysis and electron transport by 2-stepped photo-driving in photosynthesis by green plants in illustrated.

Adenosine Triphosphate↗
PubMedDate not supplied

Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase.

In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.

Antibody Specificity↗
PubMed1971

Effect of nitrogenase components from mutant and wild-type strains of Azotobacter on the dilution effect of nitrogenase.

Nitrogenase activity exhibits a dilution effect. Evidence is presented that the reason for the dilution effect is that one of the component proteins of nitrogenase is limiting in preparations of this enzyme. The limiting component appears to be the non-haem-iron-containing protein (also called fraction II, iron protein, azoferredoxin), which is equivalent to the enhancement factor for nitrogenase activity present in extracts of nitrogenaseless mutant 22R1. A mathematical function of specific activity is described that is useful in describing nitrogenase. It takes into account the dilution effect and the exponential nature of the relationship between nitrogenase activity and enzyme protein concentration.

Azotobacter↗
PubMed1979

Nitrogenase from Rhodospirillum rubrum. Relation between 'switch-off' effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios.

Nitrogenase activity of 'membrane-free' extracts, produced from nitrogen-starved Rhodospirillum rubrum to which 4 mM NH4+ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this 'switch-off/switch-on' effect and the function of the membrane component is discussed. Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein rations. The ATP/2E- values are 4-5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.

Acetylene↗
PubMed1988

Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii.

Azotobacter vinelandii was grown diazotrophically in chemostat cultures limited by sucrose, citrate, or acetate. Specific activities of cellular oxygen consumption (qO2) and nitrogenase (acetylene reduction) were measured in situ at different dilution rates (D, representing the specific growth rate mu at steady state). Sucrose-limited cultures exhibited linear relationships between qO2 and D, each of which, however, depended on the dissolved oxygen concentration in the range of 12 to 192 microM O2. From these plots, qO2 required for maintenance processes (mO2) were extrapolated. mO2 values did not increase linearly with increasing dissolved oxygen concentrations. With citrate- or acetate-limited cultures qO2 also depended on D. At 108 microM O2, however, qO2 and mO2 of the latter cultures were significantly lower than those of sucrose-limited cultures. Specific rates of acetylene reduction increased linearly with D, irrespective of the type of limitation and of the dissolved oxygen concentration (J. Kuhla and J. Oelze, Arch. Microbiol. 149:509-514, 1988). The reversible switch-off of nitrogenase activity under oxygen stress also depended on D and was independent of qO2, mO2, or the limiting substrate. Increased switch-off effects resulting from increased stress heights could be compensated for by increasing D. Since D represents not only the supply of the carbon source but also the supply of electrons and energy, the results suggest that the flux of electrons to the nitrogenase complex, rather than qO2, stabilizes nitrogenase activity against oxygen inactivation in aerobically growing A. vinelandii.

Acetates↗
PubMed1978

Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes.

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo--Fe protein showed that they arose from Fe--S clusters in the Mo--Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the --1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme--product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe--S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

Acetylene↗
PubMed1986

Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster.

The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed.

Azotobacter↗
PubMed1998

Short-term regulation of nitrogenase activity by NH4+ in Rhodobacter capsulatus: multiple in vivo nitrogenase responses to NH4+ addition.

The photosynthetic bacterium Rhodobacter capsulatus has been shown to carry out nitrogenase "switch-off," a rapid, reversible inhibition of in vivo activity. Here, we demonstrate that highly nitrogen-limited cultures of both the wild-type strain and a draT draG mutant are capable of nitrogenase switch-off while moderately nitrogen-limited cultures show instead a "magnitude" response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added NH4+.

ADP Ribose Transferases↗
PubMed1997

The first glimpse of a complex of nitrogenase component proteins by solution X-ray scattering: conformation of the electron transfer transition state complex of Klebsiella pneumoniae nitrogenase.

An essential feature of the mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, is the formation of a transient electron transfer complex between the MoFe protein containing the active site at which N2 is reduced, and the Fe protein, which functions as a specific electron donor to the MoFe protein. We have obtained high quality solution X-ray scattering data using synchrotron X-rays of a stable putative electron transfer complex, (MoFe-protein)(Fe-protein.ADP.AIF4)2, of Klebsiella pneumoniae and used the model-independent approach based on the multipole expansion method to provide a stable and unique shape restoration at approximately 15 A resolution. The biological significance of this first molecular structure of a nitrogenase complex is discussed.

Azotobacter vinelandii↗
PubMed1972

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

The electron paramagnetic resonance of nitrogenase components, separately and together with the other reactants in the nitrogenase system (namely, reductant and Mg.ATP), have been examined at low temperatures (<20 degrees K). The MoFe protein, component I or molybdoferredoxin, in the oxidized (but not oxygen-inactivated) state yields signals with g-values of 4.3, 3.7, and 2.01, and when reduced has no observable electron paramagnetic resonance. The Fe protein, component II, or azoferredoxin, yields a signal with g-values of 2.05, 1.94, and 1.89 in the reduced state that is converted by Mg.ATP into an axial signal with g-values near 2.05 and 1.94, and a second split signal near g = 4.3. The Fe protein has no definite electron paramagnetic resonance in the oxidized (not oxygen-denatured) state under these conditions. The Mg.ATP complex of reduced Fe protein reduces the MoFe protein, whereas dithionite alone does not reduce the MoFe protein. Reoxidation of the system by substrate leads to disappearance of the Fe protein signal and the reappearance of the MoFe protein signal. Thus Mg.ATP, which is hydrolyzed during substrate reduction, converts the Fe protein to a reductant capable of transferring electrons to MoFe protein, after which substrate reduction occurs.

Adenosine Triphosphate↗