Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Molybdoferredoxin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed2003

Nitrogen fixation: the mechanism of the Mo-dependent nitrogenase.

This review focuses on recent developments elucidating the mechanism of the Mo-dependent nitrogenase. This enzyme, responsible for the majority of biological nitrogen fixation, is composed of two component proteins called the MoFe protein and the Fe protein. Recent progress in understanding the mechanism of this enzyme has focused on elucidating the structures of the active site metal clusters and of the proteins, understanding substrate interactions with the active site, defining the flow of electron transfer between the metal clusters, and defining the various roles of MgATP hydrolysis.

Adenosine Triphosphate↗
PubMed1983

Biological nitrogen fixation: primary structure of the Rhizobium trifolii iron protein gene.

Biological nitrogen fixation in the Rhizobium-legume symbiosis is dependent on the induction of a bacterially-encoded enzyme complex, nitrogenase. To examine the organization and expression of the genes encoding the components of nitrogenase in this complex system, these genes have been isolated from the legume symbiont Rhizobium trifolii by molecular cloning. DNA sequence analysis of the entire nifH gene (encoding the Fe-protein component of nitrogenase) and of the amino-terminal 141 codons of the nifD gene (encoding the alpha-subunit of the Mo-Fe protein) indicates that these genes are linked on a single operon in this strain. The Fe-protein amino acid sequence shares considerable homology with the sequence from other organisms, in particular the related organism Rhizobium meliloti (90% homology). The nif structural genes are preceded by a DNA sequence which is repeated at least three times in the Rhizobium trifolii genome.

Amino Acid Sequence↗
PubMed1987

Studies on nitrate reductase of Clostridium perfringens. IV. Identification of metals, molybdenum cofactor, and iron-sulfur cluster.

Nitrate reductase of Clostridium perfringens was purified by an improved method using immuno-affinity chromatography. The purified preparation contained Mo, Fe, and acid-labile sulfide; the Mo content was 1 mol per mol and the Fe 3.7 mol per mol of the enzyme. The inactive enzyme obtained from cells grown in the presence of tungstate did not hold Mo but contained 1 mol of W. The content of Fe was not increased. The presence of molybdenum cofactor in the nitrate reductase was indicated by the formation of molybdopterin form A in the oxidation of the enzyme by iodine and by the complementation of NADPH-nitrate reductase with the heart-treated enzyme in the extract of Neurospora crassa nit-1. The Clostridium nitrate reductase had an absorption maximum at 279 nm and shoulders at 320, 380, 430, and 520 nm. This enzyme seems to contain an iron sulfur cluster since the reduced enzyme showed decreased absorption in visible region. The CD spectrum of the enzyme has a positive peak at 425 nm and negative ones at 310, 360, and 595 nm. It was compared with the CD spectrum of ferredoxin (2Fe-2S or 4Fe-4S cluster) and the nitrate reductase of Plectonema boryanum.

Chromatography, Affinity↗
PubMed1981

Correspondence of the larger subunit of the MoFe-protein in clostridial nitrogenase to the nif D gene products of other N2-fixing organisms.

The amino(N)-terminal sequence of the larger subunit (alpha) of the MoFe-protein from Clostridium pasteurianum was determined up to 179 amino acid residues by analyses of BrCN and tryptic peptides of the original subunit. Apparent similarities exist among the sequence of the clostridial alpha-subunit, that of the smaller subunit (beta) of the Azotobacter vinelandii MoFe protein, and those predicted from the nucleotide sequences of nif D genes in Klebsiella pneumoniae and Anabaena 7120. In comparing the sequences of C. pasteurianum and K. pneumoniae, 45% of residues are identical of a total of 184 sites. Therefore, the larger subunit of the clostridial MoFe-protein must correspond to the nif D gene product of K. pneumoniae.

Amino Acid Sequence↗
PubMed1987

New, man-made N2-fixing systems.

The major inputs of fixed N into the global nitrogen cycle are assessed and compared as indicators of both the need for and the likely basis of new, complementary, man-made N2-fixing processes. The development, since 1964, of the purely chemical, highly reactive systems for the reduction of N2, including those driven electro- and photochemically, is traced, along with the parallel efforts to synthesize metal-N2 complexes (the first step in any likely fixation process) and subsequently protonate them to produce hydrazine or ammonia. These experimental approaches are convergent. Successful cycling or catalysing of some of these N2-binding systems has been achieved. The advantages and limitations of the more successful systems are noted. Approaches to this problem via direct modelling of the nitrogenase active site are outlined, as is the one successful use of such complexes in achieving N2 reduction. This wealth of effort on the reductive approaches contrasts vividly with the almost complete absence of research on N2 oxidation. Currently, only a re-evaluation of the arc discharge process is continuing. Finally, the author's studies of the extruded molybdenum-containing prosthetic group of nitrogenase, the enzymic N2-reducing site, are described in relation to future N2-fixing systems.

Agriculture↗
PubMed1999

Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152.

In cultures of the unicellular cyanobacterium Gloeothece sp. ATCC 27152 growing under alternating 12 h light and 12 h darkness, nitrogenase activity appears as cultures enter the dark phase. Synthesis of both component proteins of nitrogenase commences immediately prior to the appearance of activity and continues until about 8 h into the period of darkness. The two components (Fe-protein and MoFe-protein) are synthesized in a molar ratio of about 3:1. Degradation of the nitrogenase proteins starts as early as 4 h into the dark period and increases markedly as cultures enter the light phase. As a result, both nitrogenase proteins are completely absent from cultures during most of the light phase. In contrast, all of the other proteins investigated appeared to be present throughout the cycle of alternating light and darkness. Degradation of nitrogenase depends upon protein synthesis during the last 6 h of darkness and is prevented by addition of protease inhibitors. Two proteins, of M(r) 47,000 and 29,000, are specifically synthesized during this period and it is possible that they have a role in nitrogenase degradation. Proteolytic activity of extracts of Gloeothece, measured as the ability to degrade azocasein, increased markedly during the early part of the light period, but this increase did not depend on protein synthesis. This activity does not therefore correspond to that specifically involved in nitrogenase catabolism, though it may act on initial breakdown products generated by a nitrogenase-specific degradative system. A phycobiliprotein appears to act as a temporary store of the degradation products of nitrogenase.

Autoradiography↗
PubMed2004

Mo K- and L-edge X-ray absorption spectroscopic study of the ADP.AlF4--stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal.

The utility of using X-ray absorption spectroscopy (XAS) to study metalloproteins and, specifically, the enzyme complex nitrogenase, is highlighted by this study comparing both the structural and Mo-localized electronic features of the iron-molybdenum cofactor (FeMoco) in isolated MoFe protein and in the ADP.AlF4--stabilized complex of the MoFe protein with the Fe protein. No major differences are found at Mo between the two protein forms. The excellent quality of the data at both the Mo K and L edges will provide a baseline for analysis of other intermediates in the nitrogenase cycle. A new capability to delineate various contributions in the resting state of FeMoco is being pursued through polarized single-crystal XAS. The initial results point to the feasibility of using this technique for the analysis of scattering from the as yet unidentified atom at the center of FeMoco.

Adenosine Diphosphate↗
PubMed2000

Interaction with magnesium and ADP stabilizes both components of nitrogenase from Klebsiella pneumoniae against urea denaturation.

The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild-type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2 and GDP. Stabilization by Mg+2 is slight for the holo MoFe protein (from approximately 1.5 to approximately 2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifH gene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2 alone, to approximately 3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of the nifM gene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifH gene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild-type enzyme in the absence of Mg+2 but comparable stability in its presence, showing the importance of Mg+2 in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild-type even with Mg+2 added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild-type.

Adenosine Diphosphate↗
PubMed2006

NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.

The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.

Azotobacter vinelandii↗
PubMed1985

The importance of quantitative Mössbauer spectroscopy of MoFe-protein from Azotobacter vinelandii.

The Mössbauer spectra of MoFe-protein of Azotobacter vinelandii, as isolated under dithionite and taken at temperatures from 125 K to 175 K, are the sums of four resolved quadrupole doublets. Our results indicate that the currently accepted interpretation of these doublets can be questioned. Our data reduction method converts the Mössbauer transmission spectra to source lineshape deconvolved absorption spectra linear in iron. We used these absorption spectra to determine the stoichiometry of the Fe clusters in MoFe-protein and we obtained much better fits if we assumed that there are four iron atoms in the 'Fe2+, doublet, two iron atoms in the 'S' doublet, twelve iron atoms in the 'D' doublet and sixteen iron atoms in the 'M' doublet. Therefore we propose that the MoFe-cofactor contains one molybdenum and eight iron atoms ('M'). We also argue that none of the previous Mössbauer spectroscopic studies have been performed on the highest-activity preparation now obtainable, nor has there been any study to prove that the Mössbauer spectra are independent of activity. We consider that the Mössbauer spectroscopic studies of the MoFe-protein of nitrogenase are a re-opened and unsolved problem.

Azotobacter↗
PubMed1985

Electron microscopy of the Mo-Fe-protein from Azotobacter vinelandii nitrogenase.

The quaternary structure of the Mo-Fe-protein from Azotobacter vinelandii has been studied by electron microscopy. A model of the molecule of the Mo-Fe-protein has been proposed: two alpha subunits are displaced relative to two beta subunits along a twofold axis, so the molecule can be characterized by the point-group pseudosymmetry 222. Computer averaging of the images showed that one of the projections of the molecule could be characterized by twofold rotational symmetry. Micrographs of nitrogenase recombined complex (Mo-Fe-protein + Fe-protein) have been obtained. They showed particles close in size and form to the Mo-Fe-protein molecule. Therefore, it has been proposed that the Fe-protein could be situated in the central cavity of Mo-Fe-protein.

Azotobacter↗
PubMed1986

The nifH gene product is required for the synthesis or stability of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae.

The MoFe protein of nitrogenase from Klebsiella pneumoniae contains an iron-molybdenum cofactor, FeMoco, the synthesis or processing of which involves the products of at least five genes, nifQ, nifB, nifN, nifE and nifV. We have detected FeMoco activity in extracts of strains which synthesise neither of the MoFe protein subunits, indicating that FeMoco can be synthesised prior to combination with the MoFe protein polypeptides. Expression of the nifH gene (or a large part of it), was essential for FeMoco activity to be observed either in the presence or in the absence of the MoFe protein subunits. The nifH gene product was not involved in the control of the transcription of other nif gene products known to be involved in FeMoco synthesis or processing, nor was it essential for the stability of performed FeMoco before its combination with the MoFe protein polypeptides.

Bacterial Proteins↗
PubMed1987

Quantitative EPR of an S = 7/2 system in thionine-oxidized MoFe proteins of nitrogenase. A redefinition of the P-cluster concept.

Thionine-oxidized nitrogenase MoFe proteins from Azotobacter vinelandii. Azotobacter chroococcum and Klebsiella pneumoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin Hamiltonian parameters D = -3.7 +/- 0.7 cm-1, [E] = 0.16 +/- 0.01 cm-1 and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g approximately 10, approximately 2.5 and approximately 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters. Quantification relative to the S = 3/2 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, Pa and Pb, only one of which is further oxidized with thionine resulting in the S = 7/2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.

Azotobacter↗
PubMed1993

Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii MoFe protein.

In Azotobacter vinelandii MoFe protein the oxidation of the P clusters to the S = 7/2 state is associated with a redox reaction with Em,7.5 = +90 +/- 10 mV (vs the normal hydrogen electrode), n = 1. A concomitant redox process is observed for a rhombic S = 1/2 EPR signal with g = 1.97, 1.88 and 1.68. This indicates that both S = 1/2 and S = 7/2 signals are associated with oxidized P clusters occurring as a physical mixture of spin states. The maximal intensity of the S = 1/2 and S = 7/2 signals in the mediated equilibrium redox titration is similar if not identical to that of solid-thionine-treated samples. Summation of the spin concentration of the S = 1/2 spin state (0.25 +/- 0.03 spin/alpha 2 beta 2) and the S = 7/2 spin state (1.3 +/- 0.2 spin/alpha 2 beta 2) confirms that the MoFe protein has absolutely no more than two P clusters. In spectra of enzyme fixed at potentials around -100 mV a very low-intensity g = 12 EPR signal was discovered. In parallel-mode EPR the signal sharpened and increased > 10-fold in intensity which allowed us to assign the g = 12 signal to a non-Kramers system (presumably S = 3). In contrast with the non-Kramers EPR signals of various metalloproteins and inorganic compounds, the sharp absorption-shaped g = 12 signal is not significantly broadened into zero field, implying that the zero field splitting of the non-Kramers doublet is smaller than the X-band microwave quantum. The temperature dependence of this g = 12 EPR signal indicates that it is from an excited state within the integer spin multiplet. A bell-shaped titration curve with Em,7.5 = -307 +/- 30 mV and +81 +/- 30 mV midpoint potentials is found for the g = 12 EPR signal. We propose that this signal represents an intermediate redox state of the P clusters between the diamagnetic, dithionite-reduced and the fully oxidized S = 7/2 and S = 1/2 state. Redox transitions of two electrons (-307 +/- 30 mV) and one electron (+90 +/- 10 mV) link the sequence S = 0<-->S = 3<-->(S = 7/2 and S = 1/2). We propose to name the latter paramagnetic oxidation states of the P clusters in nitrogenase POX1 and POX2, and to retain PN for the diamagnetic native redox state.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗
PubMed1993

Detection of the in vivo incorporation of a metal cluster into a protein. The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. The MoO4(2-) concentration causing 50% repression of the alternative nitrogenase in nifHDK- cells was 6 nM. If MoO4(2-) was added to a growing nifHDK- culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H4 formation decreased continuously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yield of C2H6 increased from 2% (C2H6/C2H4 x 100) in the absence of MoO4(2-), to a maximal value of 69% in the presence of MoO4(2-) (1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MoO4(2-) concentration in the medium and the longer the incubation time. In the presence of ReO4-, WO4(2-) or VO4(3-), a similar effect did not occur. The 'Mo effect' was not observed in a nifHDK- nifE- double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK- nifQ- mutant. Crude extracts from nifHDK- cells cultivated in the presence of MoO4(2-), also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase. The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis. The analysis of nifE-lacZ and nifN-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO4(2-) deficiency. The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.

Chloramphenicol↗
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↗
PubMed1997

Comparative in-vivo and in-vitro 99Mo-time-differential-perturbed-angular-correlation studies on the nitrogenase MoFe protein and on other Mo species of different N2-fixing bacteria.

Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing 99MoO4(2-) . The distribution of 99Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion-exchange (DEAE-Sephacel) chromatography. Cells of K. pneumoniae took up MoO4(2-) only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kp1) as the predominant Mo-containing species. In the case of A. vinelandii, under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Av1). However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo-storage protein. In the presence of 20 mM NH4+, conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo-storage protein exclusively. This protein proved to be unstable towards DEAE-Sephacel, apparently releasing all the molybdenum in form of MoO4(2-) during the fractionation procedure. R. capsulatus contained, in addition to the MoFe protein (Rc1), significant amounts of other not-yet-identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase. The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process 99Mo(beta-)99Tc using time differential perturbed angular correlation spectroscopy. The quadrupole coupling constant (v(Q)) determined for the Mo center in MoFe proteins was consistently in the range 66-81 MHz. The values of the coupling constants determined with intact cells and with the isolated, partially purified, MoFe proteins were in very good agreement. For the Mo-storage protein of A. vinelandii, a quadrupole coupling constant of approximately 180 MHz was determined by measurements performed with nitrogenase-repressed cells as well as with gel-filtered cell-free extracts. Our work proves that the relevant study of hyperfine interactions allows the identification of the MoFe protein and also other Mo proteins in vivo as well as in vitro.

Azotobacter vinelandii↗