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.     

Nitrogenase of Klebsiella pneumoniae. A stopped-flow study of magnesium-adenosine triphosphate-induce electron transfer between the compeonent proteins.

Stopped-flow kinetic data have been obtained for a rapid electron-transfer reaction between the component proteins of nitrogenase from Klebsiella pneumoniae, which was induced by MgATP. Up to three equivalents of the Fe-containing protein were rapidly oxidized by one equivalent of the Fe-Mo-containing protein in a unimolecular reaction, k2 = 2 x 10(2)S-1. Evidence for a tight complex between the component proteins, KD(complex) less than 0.5 muM, which was formed with a rate k1 greater than 1 x 10(7)M-1-S-1, has been obtained. MgATP bound to either the Fe-containing protein or to the two-protein complex with a rate k3 greater than 2.5 x 10(6)M-1-S-1 and with KD(MgATP) = 0.4mM, before the electron-transfer reaction.     

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.     

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.     

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.     

New insights into structure-function relationships in nitrogenase: A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein.

The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 A, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 A resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser beta90 and a surface helix in the beta subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the alpha subunit that may have a functional role. It is within 10 A of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.     

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.     

Synthesis of the iron-molybdenum cofactor of nitrogenase is inhibited by a low-molecular-weight metabolite of Klebsiella pneumoniae.

The in vitro synthesis of the iron-molybdenum cofactor nitrogenase was inhibited by a low-molecular-weight factor. This inhibitory factor was present in the membrane extracts of wild-type and nif mutant strains of Klebsiella pneumoniae that were grown under conditions that either repressed or derepressed nitrogenase expression. In vitro, the inhibition was specific for the NifB protein. Addition of this factor to K. pneumoniae cells at various times during nif derepression decreased nitrogenase activity, presumably through inhibition of iron-molybdenum cofactor synthesis. The inhibitor was purified by solvent extraction and chromatography on DEAE-cellulose, silica gel, and aluminum oxide columns.     

Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane.

Ethylene (C2H4) inhibited H2 evolution by the Mo-containing nitrogenase of Klebsiella pneumoniae. The extent of inhibition depended on the electron flux determined by the ratio of Fe protein (Kp2) to MoFe protein (Kp1) with KiC2H4 = 409 kPa ([Kp2]/[Kp1] = 22:1) and KC2H4i = 88 kPa ([Kp1]/[Kp2] = 21:1) at 23 degrees C at pH 7.4. At [Kp2]/[Kp1] = 1:1, inhibition was minimal with C2H4 (101 kPa). Extrapolation of data obtained when C2H4 was varied from 60 to 290 kPa indicates that at infinite pressure of C2H4 total inhibition of H2 evolution should occur. C2H4 inhibited concomitant S2O4(2-) oxidation to the same extent that it inhibited H2 evolution. Although other inhibitors of total electron flux such as CN- and CH3NC uncouple MgATP hydrolysis from electron transfer, C2H4 did not affect the ATP/2e ratio. Inhibition of H2 evolution by C2H4 was not relieved by CO. C2H4 was reduced to C2H6 at [Kp2]/[Kp1] ratios greater than or equal to 5:1 in a reaction that accounted for no more than 1% of the total electron flux. These data are discussed in terms of the chemistry of alkyne and alkene reduction on transition-metal centres.     

Iron K-edge X-ray absorption spectroscopy of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae.

Iron K-edge X-ray absorption data for the iron-molybdenum cofactor ('FeMoco') from Klebsiella pneumoniae reported here provide the first evidence for long-range structural order in the cofactor [Fe...Fe(Mo) = 0.368 nm in addition to Fe...S = 0.22 nm and Fe...Fe(Mo) = 0.27 nm] and, in contrast with previously published data [Antonio, Teo, Orme-Johnson, Nelson, Groh, Lindahl, Kauzlarich & Averill (1982) J. Am. Chem. Soc. 104, 4703-4705], indicate that most of the iron centres are not co-ordinated to light (oxygen, nitrogen) atoms. This demonstrates that presently available chemical models for FeMoco are inadequate.     

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.     

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe 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.