scholarly journals A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase

1989 ◽  
Vol 264 (3) ◽  
pp. 657-661 ◽  
Author(s):  
R N F Thorneley ◽  
G Ashby ◽  
J V Howarth ◽  
N C Millar ◽  
H Gutfreund
Keyword(s):  
Electron Transfer ◽  
Klebsiella Pneumoniae ◽  
Equilibrium Constant ◽  
Stopped Flow ◽  
Rabbit Muscle ◽  
Flow Calorimetry ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.

scholarly journals 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

1991 ◽  
Vol 279 (1) ◽  
pp. 81-85 ◽  
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.

Electron transfer and half-reactivity in nitrogenase

2011 ◽  
Vol 39 (1) ◽  
pp. 201-206 ◽  
Author(s):  
Thomas A. Clarke ◽  
Shirley Fairhurst ◽  
David J. Lowe ◽  
Nicholas J. Watmough ◽  
Robert R. Eady
Keyword(s):  
Electron Transfer ◽  
Protein Molecule ◽  
Stopped Flow ◽  
Protein Binding Sites ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Mofe Protein ◽  
Fe Protein ◽  
Rapid Quench ◽  
Important Enzyme

Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum–iron protein (MoFe protein) and a protein containing an [Fe4–S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

Changes in the 31P NMR spectrum of rabbit muscle myosin subfragment 1 ·MgADP with temperature

2002 ◽  
Vol 402 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Bruce D Ray ◽  
Mikhail I Khoroshev ◽  
Kathleen Ue ◽  
Manuel F Morales ◽  
B.D Nageswara Rao
Keyword(s):  
31P Nmr ◽  
Rabbit Muscle ◽  
Nmr Spectrum ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Muscle Myosin

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

1984 ◽  
Vol 224 (3) ◽  
pp. 877-886 ◽  
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.

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

1987 ◽  
Vol 247 (3) ◽  
pp. 547-554 ◽  
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.

scholarly journals Kinetics of nitrogenase of Klebsiella pneumoniae. Heterotropic interactions between magnesium-adenosine 5′-diphosphate and magnesium-adenosine 5′-triphosphate

1977 ◽  
Vol 165 (2) ◽  
pp. 255-262 ◽  
Author(s):  
R N F Thorneley ◽  
A Cornish-Bowden
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Protein A ◽  
Competitive Inhibitor ◽  
Electron Transfer Reaction ◽  
H2 Evolution ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Kinetics Of

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evulution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K′i = 20 micron was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K.pneumoniae occurred with a rate comparable with that of electron transfer, k approximately 2 × 10(2)S-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data have been fitted to symmetry and sequential-type models involving conformation changes in two identical subunits. The data suggest that the enzyme can bind up to molecules of either MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins.

scholarly journals Nitrogenases from Klebsiella pneumoniae and Clostridium pasteurianum. Kinetic investigations of cross-reactions as a probe of the enzyme mechanism

1976 ◽  
Vol 157 (2) ◽  
pp. 439-447 ◽  
Author(s):  
B E Smith ◽  
R N Thorneley ◽  
R R Eady ◽  
L E Mortenson
Keyword(s):  
Electron Transfer ◽  
Klebsiella Pneumoniae ◽  
Atp Hydrolysis ◽  
Reductase Activity ◽  
Enzyme Mechanism ◽  
Clostridium Pasteurianum ◽  
H2 Evolution ◽  
Fe Protein ◽  
Accurate Indication ◽  
Kinetic Investigations

In combination with the Mo-Fe protein of nitrogenase from Klebsiella pneumoniae, the Fe protein of nitrogenase from Clostridium pasteurianum forms an active enzyme with novel properties different from those of either of the homologous nitrogenases. The steady-state rates of reduction of acetylene and H+ are 12% of those of the homologous system from C.pasteurianim. Acetylene reductase activity exhibited an approx. 10min lag at 30 degrees C before the rate of reduction became linear, consistent with a once-only activation step being necessary for acetylene reduction to occur. No such lag was observed for H2 evolution. The activity with N2 as a reducible substrate was very low, implying that acetylene reductase activity is not necessarily an accurate indication of nitrogen-fixing ability. This is of particular relevance to studies on mutant and agronomically important organisms. Stopped-flow spectrophotometric studies showed unimolecular electron transfer from the Fe protein to the Mo-Fe protein to occur at the same rate (k2 = 2.5 × 10(2)s-1) and with the same dependence on ATP concentration (apparent KD = 400 muM) as with the homologous Klebsiella nitrogenase. However, an ATP/2e ratio of 50 was obtained for H2 evolution, indicating that ATP hydrolysis had been uncoupled from electron transfer to substrate. These data indicate that ATP has at least two roles in the mechanism of nitrogenase action. The combination of the Mo-Fe protein of nitrogenase of C.pasteurianim and the Fe protein of K.pneumoniae were inactive in all the above reactions, except for a weak adenosine triphosphatase activity, 0.5% of that of the homologous K.pneumoniae system.

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