nifV-dependent, low-molecular-weight factor required for in vitro synthesis of iron-molybdenum cofactor of nitrogenase.

The molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor of nitrogenase requires a low-molecular-weight factor. The factor is present in extracts of nitrogen fixation-derepressed cultures of Klebsiella pneumoniae and Azotobacter vinelandii, but not in extracts of repressed cultures of these bacteria. Analysis of K. pneumoniae Nif mutants has indicated that the nifV gene product is the only nif protein (besides nifA) necessary for the synthesis and accumulation of the factor. The factor is stable to oxygen, temperatures below 120 degrees C, and extremely acidic and basic conditions. The activity of the factor was completely destroyed by dry ashing or digestion with sulfuric acid. The factor has been partially purified by filtration through an Amicon PM-10 DIAFLO membrane and chromatography on DEAE-cellulose, hydroxylapatite, silica gel, and Sephadex G-25.     

Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the iron-molybdenum cofactor

The in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires homocitrate (2-hydroxy-1,2,4-butanetricarboxylic acid). Homocitrate is apparently synthesized by the nifV gene product. In the absence of homocitrate, no FeMo-co is formed in vitro, as determined from coupled C2H2 reduction assays and the lack of 99Mo label incorporation into apodinitrogenase. Several organic acids were tested for their ability to replace homocitrate in the FeMo-co synthesis system. With appropriate homocitrate analogues, aberrant forms of FeMo-co are synthesized that exhibit altered substrate specificity and inhibitor susceptibility. Homoisocitrate (1-hydroxy-1,2,4-butanetricarboxylic acid) and 2-oxoglutarate facilitated the incorporation of 99Mo into apodinitrogenase in the FeMo-co synthesis system, yielding a dinitrogenase that effectively catalyzed the reduction of protons but not C2H2 or N2. Citrate also promoted the incorporation of 99Mo into apodinitrogenase, and the resulting holodinitrogenase reduced protons and C2H2 effectively but not N2. In addition, proton reduction from this enzyme was inhibited by CO. The properties of the homodinitrogenase formed in the presence of citrate were reminiscent of those of the Klebsiella pneumoniae NifV- dinitrogenase. We also observed low rates of HD formation from NifV- dinitrogenase compared to those from the wild-type enzyme. No HD formation was observed with the dinitrogenase activated in vitro in the presence of citrate. We propose that in vivo NifV- mutants utilize citrate for FeMo-co synthesis.     

Chromomycin dimer-DNA oligomer complexes. Sequence selectivity and divalent cation specificity

This paper reports on a solution NMR characterization of the sequence selectivity and metal ion specificity in chromomycin-DNA oligomer complexes in the presence of divalent cations. The sequence selectivity studies have focused on chromomycin complexes with the self-complementary d(A1-A2-G3-G4-C5-C6-T7-T8) duplex containing a pair of adjacent (G3-G4).(C5-C6) steps and the self-complementary d(A1-G2-G3-A4-T5-C6-C7-T8) duplex containing a pair of separated (G2-G3).(C6-C7) steps in aqueous solution. The antitumor agent (chromomycin) and nucleic acid protons have been assigned following analysis of distance connectivities in NOESY spectra and coupling connectivities in DQF-COSY spectra for both complexes in H2O and D2O solution. The observed intermolecular NOEs establish that chromomycin binds as a Mg(II)-coordinated dimer [1 Mg(II) per complex] and contacts the minor-groove edge with retention of 2-fold symmetry centered about the (G3-G4-C5-C6).(G3-G4-C5-C6) segment of the d(A2G2C2T2) duplex. By contrast, complex formation is centered about the (G2-G3-A4-T5).(A4-T5-C6-C7) segment and results in removal of the two fold symmetry of the d(AG2ATC2T) duplex. Thus, the binding of one subunit of the chromomycin dimer at its preferred (G-G).(C-C) site assists in the binding of the second subunit to the less preferred adjacent (A-T).(A-T) site. These observations suggest a hierarchy of chromomycin binding sites, with a strong site detected at the (G-G) step due to the hydrogen-bonding potential of acceptor N3 and donor NH2 groups of guanosine that line the minor groove. The divalent cation specificity has been investigated by studies on the symmetric chromomycin-d(A2G2C2T2) complex in the presence of diamagnetic Mg(II), Zn(II), and Cd(II) cations and paramagnetic Ni(II) and Co(II) cations. A comparative NOESY study of the Mg(II) and Ni(II) symmetric complexes suggests that a single tightly bound divalent cation aligns the two chromomycins in the dimer through coordination to the C1 carbonyl and C9 enolate ions on the hydrophilic edge of each aglycon ring. Secondary divalent cation binding sites involve coordination to the major-groove N7 atoms on adjacent guanosines in G-G steps. This coordination is perturbed on lowering the pH below 6.0, presumably due to protonation of the N7 atoms. The midpoint of the thermal dissociation of the symmetric complex is dependent on the divalent cation with the stability for reversible transitions decreasing in the order Mg(II) greater than Zn(II) greater than Cd(II) complexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis of iron-molybdenum cofactor in the absence of nitrogenase.

Klebsiella pneumoniae accumulates molybdenum during nitrogenase derepression. The molybdenum is primarily in nitrogenase component I in the form of iron-molybdenum cofactor (FeMo-co). Mutations in any of three genes (nifB, nifN, and nifE) involved in the biosynthesis of FeMo-co resulted in very low molybdenum accumulation and in a molybdenum-free nitrogenase component I. A mutant lacking both subunits of nitrogenase component I accumulated 60% of the amount of molybdenum present in the wild type. The molybdenum was in protein-bound form and behaved differently than that in the wild type with respect to electrophoretic mobility, size, and extractability by organic solvents. Two forms of molybdenum could be extracted from the protein fraction of the mutant; one of them was not detected in the wild type, and the other behaved like FeMo-co in nonaqueous gel filtration chromatography. Crude extracts of this mutant were able to complement in vitro K. pneumoniae or Azotobacter vinelandii mutants unable to produce FeMo-co. These data show that biosynthesis of FeMo-co does not require the presence of nitrogenase component I. In its absence, FeMo-co is accumulated on a different protein, presumably an intermediate in the normal FeMo-co biosynthetic pathway.     

Biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a Mo-Fe-S cluster that has been proposed as the site of substrate reduction for the nitrogenase enzyme complex. Biosynthesis of FeMo-co in Klebsiella pneumoniae requires at least six nif (nitrogen fixation) gene products. One of the nif genes, nifV, apparently encodes a homocitrate synthase. The synthesis and accumulation of homocitrate [(R)-2-hydroxy-1,2,4-butanetricarboxylic acid] in K.pneumoniae is correlated to the presence of a functional nifV gene. K.pneumoniae strains with mutations in nifV synthesize and accumulate an aberrant form of FeMo-co. Nitrogenase from NifV- mutants is capable of reducing some of the substrates of nitrogenase effectively (e.g. acetylene), but reduces N2 poorly. With the aid of an in vitro FeMo-co synthesis system, it recently has been established that homocitrate is an endogenous component of FeMo-co. Substitution of homocitrate with other carboxylic acids results in the formation of aberrant forms of FeMo-co with altered substrate reduction capability.     

Nucleotide and bivalent cation specificity of the insulin-granule proton translocase

3-(4-[(3-Chlorophenyl)methoxy]phenyl)-5-[(methylamino)methyl]-2- oxazolidinone methanesulphonate (compound MD 780236) is a selective inhibitor of the B-form of monoamine oxidase. Inhibition involves an initial non-covalent interaction between enzyme and inhibitor followed by a time-dependent process resulting in irreversible inhibition. The initial, reversible, phase of inhibition was found to be competitive with respect to phenethylamine and 5-hydroxytryptamine, and a comparison of the Ki values indicated the affinity of the inhibitor for the B-form of the enzyme to be some 7-fold greater than its affinity for the A-form. This selectivity was considerably enhanced by preincubation of the enzyme and inhibitor. Time courses showed that complete inhibition was not achieved under conditions where the inhibitor concentration was over 100-fold greater than that of the enzyme. Assay of the activity of monoamine oxidase by determining the release of hydrogen peroxide fluorometrically showed compound MD 780236 to be a substrate for, as well as an inhibitor of, monoamine oxidase, and kinetic analysis revealed that the rate of product formation was some 530-fold greater than that of the process leading to irreversible inhibition of the B-form of the enzyme.     

Incorporation of molybdenum into the iron-molybdenum cofactor of nitrogenase.

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide.     

Nucleotide specificity in microtubule assembly in vitro

A procedure is described for removing most of the GDP bound at the exchangeable GTP binding site (E site) of tubulin. Microtubule protein containing substoichiometric amounts of GDP at the E site is found to polymerize in response to: (a) two nonhydrolyzable ATP analogues, adenylyl imidodiphosphate (AMP-PNP) and adenylyl beta, gamma-methylenediphosphonate (AMP-PCP); and (b) substoichiometric levels of GTP or dGTP. The results are interpreted as suggesting that: (1) when GDP is removed from tubulin, the E site shows broad specificity for nucleoside triphosphates: (2) microtubule assembly can be induced by the binding of substoichiometric amounts of nucleoside triphosphate to the E site.     

Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source.

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.     

Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.     

Substrate reduction properties of dinitrogenase activated in vitro are dependent upon the presence of homocitrate or its analogues during iron-molybdenum cofactor synthesis

(R)-2-Hydroxy-1,2,4-butanetricarboxylic acid [(R)-homocitrate] has been has been recently reported to be an integral constituent of the otherwise thought to be inorganic iron-molybdenum cofactor of dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V.K. (1989) Biochemistry 28,2768-2771]. Different organic acids can substitute for homocitrate in an in vitro system for iron-molybdenum cofactor synthesis and incorporation into dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V. K. (1988) Biochemistry 27, 3647-3652]. Dinitrogenase activated with homocitrate-FeMo-co was able to reduce dinitrogen, acetylene, and protons efficiently. Homoisocitrate and isocitrate dinitrogenases did not reduce dinitrogen or acetylene, but showed very high proton reduction activities. Citrate and citramalate dinitrogenases had very low dinitrogen reduction activities and intermediate acetylene and proton reduction activities. CO inhibited proton reduction in both these cases but not in the case of dinitrogenases activated with other homocitrate analogues. By use of these and other commercially available homocitrate analogues in the in vitro system, the structural features of the homocitrate molecule absolutely required for the synthesis of a catalytically competent iron-molybdenum cofactor were determined to be the hydroxyl group, the 1- and 2-carboxyl groups, and the R configuration of the chiral center. The stringency of the structural requirements was dependent on the nitrogenase substrate used for the assay, with dinitrogen having the most stringent requirements followed by acetylene and protons.     

Oxidation-reduction properties and complexation reactions of the iron-molybdenum cofactor of nitrogenase

The interactions of the iron-molybdenum cofactor, FeMoco, isolated from acid-treated Azotobacter vinelandii molybdenum-iron protein (Av1) with EDTA and thiophenol in N-methylformamide solution have been reinvestigated. Our studies show that EDTA alone is sufficient to eliminate the EPR signal of dithionite-reduced FeMoco. Neither light/5-deazaflavin nor carbon monoxide are required, which implies that this EPR-silent form of FeMoco does not correspond to the EPR-silent, substrate-reducing state of Av1. As EDTA-treated FeMoco does not regain EPR activity on addition of sodium dithionite or thiophenol, it is apparently distinct from the EPR-silent form of either dye-oxidized FeMoco or dye-oxidized Av1. Thiophenol sharpens the EPR signal of dithionite-reduced FeMoco and shifts the g = 3.3 feature to g = 3.6. This shift is complete at 1:1 ratio of thiophenol/Mo atom, while the EDTA effect requires about 40 molecules/Mo atom. Thiophenol and EDTA probably affect different sites of FeMoco. The binding of either reactant does not affect the activity of FeMoco as measured by its ability to reconstitute extracts of A. vinelandii mutant UW45.     

Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase

When apodinitrogenase (lacking FeMo-co) was activated with FeMo-co synthesized in vitro in the presence of 3H-labeled homocitrate, label was incorporated into dinitrogenase. The physical association of the label with FeMo-co was demonstrated by reisolation and purification of the cofactor from dinitrogenase. The presence of homocitrate in FeMo-co was established by NMR analysis of the organic acid extracted from dinitrogenase. Quantitation of homocitrate in dinitrogenase showed it to be present at a 1:1 ratio with molybdenum.     

Cyanide and methylisocyanide binding to the isolated iron-molybdenum cofactor of nitrogenase

19F NMR and x-ray absorption experiments have been performed with both the isolated FeMo cofactor and the MoFe protein of nitrogenase in search of direct evidence for substrate or inhibitor binding. Using 19F NMR as a probe and p-CF3C6H4S- as the receptor ligand, the data show that the nitrogenase inhibitors CN- and CH3NC bind to the isolated FeMo cofactor-RFS- complex in N-methylformamide with a finite formation constant. Their binding increases the electronic relaxation time of the complex and increases the life-time of the FeMo cofactor-p-CF3C6H4S- bond, Parallel molybdenum K edge and extended x-ray absorption fine structure experiments show that CH3NC does not bind to molybdenum. Although CO and N3- both relieve CN- and CH3NC inhibition of electron flow through nitrogenase, unlike the latter, they do not appear to bind to isolated FeMo cofactor. In experiments with the dithionite-reduced MoFe protein, we did not detect any changes in the molybdenum K edge or extended x-ray absorption fine structure spectra upon addition of CO, N2, C2H2, NaCN, CH3NC, or azide demonstrating that either these substrates and inhibitors do not bind to molybdenum or that the FeMo cofactor site of nitrogenase is inaccessible to substrate binding except under turnover conditions.     

sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence

Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements.     

Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca²⁺ overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca²⁺-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules <800 Da to enter the cell. The cells then lyse not by rupture of the plasmalemma but through the activation of a "death" channel that lets large proteins (e.g., 140–160 kDa) leave the cell. These changes in permeability are accompanied by the formation of membrane blebs. In this study, we took advantage of the well-known differences in affinity of various Ca²⁺-binding proteins for Ca²⁺ and Sr²⁺ vs. Ba²⁺ to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single-cell level (time-lapse videomicroscopy), we found that the replacement of Ca²⁺ with either Sr²⁺ or Ba²⁺ delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated green fluorescent protein. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba²⁺ not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca²⁺-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba²⁺. Taken together, these results suggest that intracellular high-affinity Ca²⁺-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise. necrosis; vital dyes; membrane blebs; time-lapse videomicroscopy; fura 2     

Inhibition of iron-molybdenum cofactor binding to component I of nitrogenase

Tetrathiomolybdate inhibits iron-molybdenum cofactor (FeMo cofactor) binding to component I of nitrogenase. Molybdenum-iron cluster (a subcomponent of FeMo cofactor) and tetrathiomolybdate inhibited FeMo cofactor activation of inactive nitrogenase component I in extracts of Azotobacter vinelandii and Klebsiella pneumoniae mutant strains defective in the biosynthesis of FeMo cofactor. Addition of tetrathiotungstate, the tungsten analog of tetrathiomolybdate, to the mutant extracts had no significant inhibitory effect on subsequent activation by FeMo cofactor.