Nitrogenase of Klebsiella pneumoniae nifV mutants. Investigation of the novel carbon monoxide-sensitivity of hydrogen evolution by the mutant enzyme

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.

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Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor

Biochemical Journal ◽

10.1042/bj2170317 ◽

1984 ◽

Vol 217 (1) ◽

pp. 317-321 ◽

Cited By ~ 110

Author(s):

T R Hawkes ◽

P A McLean ◽

B E Smith

Keyword(s):

Klebsiella Pneumoniae ◽

Active Site ◽

Molybdenum Cofactor ◽

Wild Type ◽

H2 Evolution ◽

Mofe Protein ◽

Fe Protein ◽

Metal Contents ◽

Altered Form ◽

Iron Molybdenum Cofactor

When the iron-molybdenum cofactor (FeMoco) was extracted from the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae and combined with the FeMoco-deficient MoFe protein from a nifB mutant, the resultant MoFe protein exhibited the NifV phenotype, i.e. in combination with wild-type Fe protein it exhibited poor N2-fixation activity and its H2-evolution activity was inhibited by CO. These data provide strong evidence that FeMoco contains the active site of nitrogenase. The metal contents and e.p.r. properties of FeMoco from wild-type and nifV mutants of K. pneumoniae are very similar.

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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

Biochemical Journal ◽

10.1042/bj2240895 ◽

1984 ◽

Vol 224 (3) ◽

pp. 895-901 ◽

Cited By ~ 77

Author(s):

D J Lowe ◽

R N F Thorneley

Keyword(s):

Klebsiella Pneumoniae ◽

Rate Constants ◽

Competitive Inhibitor ◽

H2 Evolution ◽

Protein Ratio ◽

Mofe Protein ◽

Fe Protein ◽

Mutual Displacement ◽

Kinetics Of

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.

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

Biochemical Journal ◽

10.1042/bj2240877 ◽

1984 ◽

Vol 224 (3) ◽

pp. 877-886 ◽

Cited By ~ 107

Author(s):

D J Lowe ◽

R N Thorneley

Keyword(s):

Steady State ◽

Klebsiella Pneumoniae ◽

Necessary Condition ◽

H2 Evolution ◽

Mofe Protein ◽

Fe Protein ◽

Steady State Kinetics ◽

Rapid Quench ◽

Steady State Kinetic ◽

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.

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

Biochemical Journal ◽

10.1042/bj2470547 ◽

1987 ◽

Vol 247 (3) ◽

pp. 547-554 ◽

Cited By ~ 23

Author(s):

G A Ashby ◽

M J Dilworth ◽

R N F Thorneley

Keyword(s):

Electron Transfer ◽

Transition Metal ◽

Klebsiella Pneumoniae ◽

Electron Flux ◽

H2 Evolution ◽

Total Electron ◽

Mofe Protein ◽

Fe Protein ◽

Total Inhibition ◽

Nitrogenase Inhibition

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.

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Reduction of cyclopropene by NifV- and wild-type nitrogenases from Klebsiella pneumoniae

Biochemical Journal ◽

10.1042/bj2580487 ◽

1989 ◽

Vol 258 (2) ◽

pp. 487-491 ◽

Cited By ~ 8

Author(s):

J P Gemoets ◽

M Bravo ◽

C E McKenna ◽

G J Leigh ◽

B E Smith

Keyword(s):

Klebsiella Pneumoniae ◽

Azotobacter Vinelandii ◽

Electron Flux ◽

H2 Production ◽

Relative Increase ◽

Analogous Reaction ◽

Wild Type ◽

H2 Evolution ◽

Product Ratio ◽

In The Wild

The nitrogenase from wild-type Klebsiella pneumoniae reduces cyclopropene to cyclopropane and propene in the ratio 1:2 at pH 7.5. We show in this paper that the nitrogenase from a nifV mutant of K. pneumoniae also reduces cyclopropene to cyclopropane and propene, but the ratio of products is now 1:1.4. However, both nitrogenases exhibit the same Km for cyclopropene (2.1 x 10(4) +/- 0.2 x 10(4) Pa), considerably more than the Km for the analogous reaction with Azotobacter vinelandii nitrogenase under the same conditions (5.1 x 10(3) Pa). Analysis of the data shows that the different product ratio arises from the slower production of propene compared with cyclopropane by the mutant nitrogenase. During turnover, both nitrogenases use a large proportion of the electron flux for H2 production. CO inhibits the reduction of cyclopropene by both K. pneumoniae proteins, but the mutant nitrogenase exhibits 50% inhibition at approx. 10 Pa, whereas the corresponding value for the wild-type nitrogenase is approx. 110 Pa. However, H2 evolution by the mutant enzyme is much less affected than is cyclopropene reduction. CO inhibition of cyclopropene reduction by the nitrogenases coincides with a relative increase in H2 evolution, so that in the wild-type (but not the mutant) the electron flux is approximately maintained. The cyclopropane/propene production ratios are little affected by the presence of CO within the pressure ranges studied at least up to 50% inhibition.

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Nitrogenase of Klebsiella pneumoniae. Distinction between proton-reducing and acetylene-reducing forms of the enzyme: effect of temperature and component protein ratio on substrate-reduction kinetics

Biochemical Journal ◽

10.1042/bj1670457 ◽

1977 ◽

Vol 167 (2) ◽

pp. 457-461 ◽

Cited By ~ 20

Author(s):

R N F Thorneley ◽

R R Eady

Keyword(s):

Klebsiella Pneumoniae ◽

Acetylene Reduction ◽

Electron Flow ◽

Turnover Time ◽

Effect Of Temperature ◽

Lag Phase ◽

H2 Evolution ◽

Protein Ratio ◽

Substrate Reduction ◽

Fe Protein

Non-linear rates of acetylene reduction and concomitant H2 evolution were observed for the nitrogenase of Klebsiella pneumoniae at 10 degrees C. A lag phase of 1-4 min, dependent on the ratio of Mo-Fe protein to Fe protein present, occurred before linear rates of acetylene reduction were achieved. A complementary burst phase for concomitant H2 evolution in the presence of acetylene was also observed. When the proton was the only reducible substrate present, linear rates of H2 evolution were observed. N2 was a poor substrate under these conditions. Similar lag and burst phases occurred at 30 degrees C, but only when a large molar excess of Mo-Fe protein with respect to Fe protein was present. The results at 10 degrees C show that the binding of acetylene to the enzyme stimulates electron flow, but that these electrons, which initially reduce protons, can only reduce acetylene after a lag phase that cannot be accommodated in the turnover time calculated under steady-state conditions.

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Klebsiella pneumoniae nitrogenase. The pre-steady-state kinetics of MoFe-protein reduction and hydrogen evolution under conditions of limiting electron flux show that the rates of association with the Fe-protein and electron transfer are independent of the oxidation level of the MoFe-protein

Biochemical Journal ◽

10.1042/bj2790081 ◽

1991 ◽

Vol 279 (1) ◽

pp. 81-85 ◽

Cited By ~ 18

Author(s):

K Fisher ◽

D J Lowe ◽

R N F Thorneley

Keyword(s):

Electron Transfer ◽

Steady State ◽

Klebsiella Pneumoniae ◽

Electron Flux ◽

High Electron ◽

H2 Evolution ◽

Mofe Protein ◽

Fe Protein ◽

Steady State Kinetics ◽

Kinetics Of

The pre-steady-state kinetics of H2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23 degrees C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (kobs. = 140 s-1). These data support our previous assumption that the rate constants that define the ‘Fe-protein cycle’ are independent of the level of reduction of the MoFe-protein.

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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

Biochemical Journal ◽

10.1042/bj2240903 ◽

1984 ◽

Vol 224 (3) ◽

pp. 903-909 ◽

Cited By ~ 68

Author(s):

R N F Thorneley ◽

D J Lowe

Keyword(s):

Klebsiella Pneumoniae ◽

Protein Concentration ◽

Dilution Effect ◽

Sodium Dithionite ◽

Evolution Rate ◽

H2 Evolution ◽

Action Simulation ◽

Mofe Protein ◽

Fe Protein ◽

Absolute Concentrations

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.

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