The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation.

The reduction of N2 to 2NH3 by Klebsiella pneumoniae nitrogenase was studied by a rapid-quench technique. The pre-steady-state time course for N2H4, formed on quenching by the acid-induced hydrolysis of an enzyme-bound intermediate in N2 reduction, showed a 230 ms lag followed by a damped oscillatory approach to a constant concentration in the steady state. The pre-steady-state time course for NH3 formation exhibited a lag of 500 ms and a burst phase that was essentially complete at 1.5s, before a steady-state rate was achieved. These time courses have been simulated by using a previously described kinetic model for the mechanism of nitrogenase action [Lowe & Thorneley (1984) Biochem. J. 224, 877-886]. A hydrazido(2-) structure (=N-NH2) is favoured for the intermediate that yields N2H4 on quenching. The NH3-formation data indicate enzyme-bound metallo-nitrido (identical to N) or -imido (=NH) intermediates formed after N-N bond cleavage to produce the first molecule of NH3 and which subsequently give the second molecule of NH3 by hydrolysis on quenching. The simulations require stoichiometric reduction of one N2 molecule at each Mo and the displacement of one H2 when N2 binds to the MoFe protein. Inhibition by H2 of N2-reduction activity occurs before the formation of the proposed hydrazido(2-) species, and is explained by H2 displacement of N2 at the active site.     

The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution.

Kinetic data for Klebsiella pneumoniae nitrogenase were used to determine the values of nine of the 17 rate constants that define the scheme for nitrogenase action described by Lowe & Thorneley [(1984) Biochem. J. 224, 877-886]. Stopped-flow spectrophotometric monitoring of the MgATP-induced oxidation of the Fe protein (Kp2) by the MoFe protein (Kp1) was used to determine the rates of association (k+1) and dissociation (k-1) of reduced Kp2(MgATP)2 with Kp1. The dependences of the apparent KNm2 on Fe protein/MoFe protein ratio and H2 partial pressure were used to determine the mutual displacement rates of N2 and H2 (k+10, k-10, k+11 and k-11). These data also allowed the rate constants for H2 evolution from progressively more reduced forms of Kp1 to be determined (k+7, k+8 and k+9). A mechanism for N2-dependent catalysis of 1H2H formation from 2H2 that requires H2 to be a competitive inhibitor of N2 reduction is also presented.     

The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration.

The rate constants from Table 1 and Scheme 2 of Lowe & Thorneley [(1984) Biochem. J. 224, 877-886] were used to simulate the rate of H2 evolution, under various conditions, from nitrogenase isolated from Klebsiella pneumoniae. These rates depend on both the ratio and concentrations of the MoFe protein and Fe protein that comprise nitrogenase. The simulations explain the shapes of 'protein titration' and 'dilution effect' curves. The concept of an apparent Km for the reductant Na2S2O4 is shown to be invalid, since the dependence of H2-evolution rate on the square root of S2O4(2-) concentration is not hyperbolic and depends on the ratio and absolute concentrations of the MoFe protein and Fe protein.     

N2O reduction and HD formation by nitrogenase from a nifV mutant of Klebsiella pneumoniae.

Dinitrogenase from a nifV mutant of Klebsiella pneumoniae contains an altered form of iron-molybdenum cofactor (FeMoco) that lacks a biologically active homocitric acid molecule. Change in the composition of FeMoco led to substantial variation in the kinetics of nitrogenase action. The KmS of the mutant enzyme for N2 and N2O were 0.244 and 0.175 atm (24,714 and 17,726 kPa), respectively. The km for N2 was higher and the Km for N2O was lower than that for the wild-type enzyme. The mutant enzyme was ineffective in N2 fixation, in N2O reduction, and in HD formation, as indicated by the low Vmax of these reactions with saturating levels of substrate and under conditions of saturating electron flux. These observations provide further support for the concept that N2, N2O, and D2 interact with the same form of dinitrogenase. H2 evolution by the mutant enzyme is only partially inhibited by CO. Observation that different numbers of electrons are stored in CO-inhibited than in noninhibited dinitrogenase before H2 is released suggests that the mutant enzyme has more sites responsible for H2 evolution than the wild-type enzyme, whose H2 evolution is not inhibited by CO.     

Klebsiella pneumoniae nitrogenase: formation and stability of putative beryllium fluoride-ADP transition state complexes.

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1.{Kp2.(MgADP.BeFx)2}2. At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1.Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations of the components allowing constants for their formation of 2.1 x 10(6) and 5.6 x 10(5) M(-1) to be calculated for the 1:1 and 2:1 complexes, respectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.     

Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae.

Nitrogenase biosynthesis in Klebsiella pneumoniae including mutant strains, which produce nitrogenase in the presence of NH+4 (Shanmugam, K.T., Chan, Irene, and Morandi, C. (1975) Biochim. Biophys. Acta 408, 101--111) is repressed by a mixture of L-amino acids. Biochemical analysis shows that glutamine synthetase activity in strains SK-24, SK-28, and SK-29 is also repressed by amino acids, with no detectable effect on glutamate dehydrogenase. Among the various amino acids, L-glutamine in combination with L-aspartate was found to repress nitrogenase biosynthesis completely. In the presence of high concentrations of glutamine (1 mg/ml) even NH+4 repressed nitrogenase biosynthesis in the strains SK-27, SK-37, SK-55 and SK-56. Under these conditions, increased glutamate dehydrogenase activity was also detected. Physiological studies show that nitrogenase derepressed strains are unable to utilize NH+4 as sole source of nitrogen for biosynthesis of glutamate for biosynthesis of glutamate, whereas back mutations leading to NH+4 utilization results in sensitivity to repression by NH+4. These findings suggest that amino acids play an important role as regulators of nitrogen fixation.     

Studies by electron paramagnetic resonance on the catalytic mechanism of nitrogenase of Klebsiella pneumoniae

The properties and catalytic reactions of the enzyme nitrogenase purified from Klebsiella pneumoniae were studied by electron-paramagnetic-resonance (e.p.r.) spectroscopy at temperatures down to 8 degrees K. The two protein fractions, Kp1 (the iron-molybdenum protein) and Kp2 (the iron protein), were examined alone and in steady-state mixtures and also in pre-steady-state experiments, by using the rapid-freezing method. Kp1 protein in dithionite solution shows a rhombic type of spectrum with g(1) 4.32, g(2) 3.63, g(3) 2.009 at pH6.8 (0 degrees C). Small changes in the spectrum produced by protons (pK=8.7 at 0 degrees C) or by acetylene indicate binding of these oxidizing substrates to this protein fraction. Kp2 protein shows a rhombic spectrum with g(1) 2.053, g(2) 1.942, g(3) 1.865, which integrates to about 0.45 electron/molecule. Binding of ATP, with a dissociation constant of 4x10(-4)m, changes the spectrum to an axial form with g( parallel) 2.036, g( perpendicular) 1.929, thus indicating a conformation change of Kp2 protein. The Kp2 protein spectrum disappears reversibly on cautious oxidation. The signals of both proteins are diminished in their steady-state mixtures, obtained in the presence of ATP and dithionite (with an ATP-generating system and Mg(2+) ions) and with protons, N(2) or acetylene as oxidizing substrate. At the same time as dithionite is consumed in such reactions, the Kp1 protein signal is gradually restored and the Kp2 protein signal diminishes to zero. In rapid-freezing experiments the signals from the two proteins decreased at indistinguishable rates (t((1/2)) about 10ms), then they remained constant. Results are interpreted in terms of a scheme in which reducing equivalents pass from dithionite to Kp2 protein, then, in an ATP-dependent reaction to Kp1 protein, this being finally reoxidized by N(2) or another oxidizing substrate. In this scheme Kp1 protein cycles between its signal-giving state and a very highly reduced signal-free state.     

Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli

A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K peptides as the substrate for the processing to form the apoMoFe protein. We also find that nifM, the gene which processes the nifH protein into Fe protein (Howard, K.S., McLean, P.A., Hansen, F. B., Lemley, P.V., Kobla, K.S. & Orme-Johnson, W.H. (1986) J. Biol. Chem. 261, 772-778) can, under certain circumstances, partially replace other processing genes (i.e. nifTYU and/or WZ) although it is not essential for apoMoFe protein formation. It also appears that nifS and nifU, reported to play a role in Fe protein production in Azotobacter vinelandii, play no such role in K. pneumoniae, although these genes are involved in apoMoFe formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature sensitivity of the regulation of nitrogenase synthesis by Klebsiella pneumoniae.

Temperature sensitivity of the regulatory protein coded by nifA prevents the organism from utilizing N2 at 37 degrees C. The purpling of 6-cyanopurine, a function of nifA expression, also is thermolabile.     

The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ''mapping''.

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide 'maps' after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.     

Pre-steady-state kinetics of intermediate formation in the deuteroferriheme-hydrogen peroxide system.

The pH dependence of formation of a peroxidatic intermediate from the reaction of deuteroferriheme with hydrogen peroxide has been determined for the region pH 8.7-10.1 from stopped-flow kinetic studies in which absorbancy changes are observed at heme monomer-dimer isosbestic points. Results are interpreted primarily in terms of the attainment of double "steady-state" concentrations of Michaelis-Menten complex I and peroxidatic intermediate I'. A linear correlation of observed first-order rate constants with alpha, the degree of dissociation of heme dimer, has been demonstrated and nonzero intercepts are obtained. Slopes and intercepts show a linear logarithmic dependence on pH which is interpreted in terms of HO2-participation both in the formation and subsequent (catalatic) decomposition of a peroxidatically active intermediate. General acid catalysis of intermediate formation is indicated from studies in phosphate, arsenate, and citrate buffer at pH 7.4-9.3. It is suggested that such catalysis may be responsible for anomalously high rates of H2O2 decomposition previously observed in phosphate buffer solution.     

Energetics of biological nitrogen fixation: determination of the ratio of formation of H2 to NH4+ catalysed by nitrogenase of Klebsiella pneumoniae in vivo.

Nitrogen fixation (Nif)-derepressed mutants of Klebsiella pneumoniae consumed, under optimum conditions, 7.5 to 8.5 mol glucose per mol N2 fixed. The nitrogenase system of these mutants catalysed the production of about 1.3 mol H2 per mol N2 reduced. Almost one-third of the energy as ATP and reductant used by nitrogenase in vivo may be lost in H2 production, since an ATP/2e ratio of approximately 4 was obtained. Nitrogenase-catalysed H2 production was not substantially suppressed by increasing the partial pressure of N2 from 0.2 atm (20 kPa) to 1 atm (101 kPa). In the absence of N2, H2 production catalysed by nitrogenase increased about threefold. It is concluded that nitrogenase-catalysed H2 production is of major importance in the overall efficiency of biological N2 fixation in vivo.     

Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae

Yoch, D. C. (South Dakota State University, Brookings), and R. M. Pengra. Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92:618-622. 1966.-The effect of exogenous amino acids and the free amino acid pool on the synthesis of the nitrogenase system of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes M5al) was investigated. When an actively N(2)-fixing culture was used to inoculate a medium containing a limiting concentration of NH(4) (+), an induction lag period was observed. When either a single amino acid or a mixture of amino acids was substituted at the same nitrogen concentration, growth was uninterrupted by the induction period. It appears that a step or steps in the formation of the nitrogenase system are repressed by NH(4) (+) and are not affected by amino acid N. The amino acids, far from repressing formation of nitrogenase as does NH(4) (+), actually stimulate its formation. It appears that both free and amino nitrogen are used simultaneously. The amino acids that served concomitantly with N(2) as a source of nitrogen were: aspartic acid, serine, threonine, leucine, and histidine. Of these amino acids, it was shown that aspartic acid is readily taken up by the cells. Of the amino acids not serving as an immediate nitrogen source, isoleucine is not taken up by the cells. The free amino acid pool of the cells was measured at the onset and termination of the induction period. Ninhydrin-positive material in the amino acid pool was depleted by 35% during the induction period.     

Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase activity.

Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase and nitrate reductase activity were characterized. The effects of mol mutations on nitrogenase activity were very similar to those caused by nifQ mutations. Mol- mutants of K. pneumoniae appear to be equivalent to ChlD- mutants of Escherichia coli.     

Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide.

Spontaneous decomposition of 5-phosphoribosyl 1-pyrophosphate at pH 5.5 was established to occur as follows: 5-Phosphoribosyl 1-pyrophosphate----5-phosphoribosyl 1,2-(cyclic)phosphate----ribose 1-phosphate----ribose Enzymic degradation of 5-phosphoribosyl 1-pyrophosphate by alkaline phosphatase from calf intestine and by acid phosphatases from potato and Aspergillus niger was found to proceed according to this pathway within the pH range 2.5-7.4 with accumulation of ribose 1-phosphate. In the case of alkaline phosphatase, Mg2+ ions inhibit the pyrophosphorolysis of 5-phosphoribosyl 1-pyrophosphate and stimulate the hydrolysis of ribose 1-phosphate.