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.

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 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 A Mössbauer spectroscopic investigation of the redox behaviour of the molybdenum-iron protein from Klebsiella pneumoniae nitrogenase. Mechanistic and structural implications

1980 ◽  
Vol 191 (2) ◽  
pp. 449-455 ◽  
Author(s):  
B E Smith ◽  
M J O'Donnell ◽  
G Lang ◽  
K Spartalian
Keyword(s):  
Klebsiella Pneumoniae ◽  
Redox Properties ◽  
Redox Process ◽  
Published Data ◽  
Hydrogen Electrode ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Fe Protein ◽  
Enzyme Turnover ◽  
Mössbauer Parameters

The redox properties of the nitrogenase Mo-Fe protein from Klebsiella pneumoniae have been monitored by 57Fe Mössbauer spectroscopy between -460 and -160mV (relative to the normal hydrogen electrode). Two redox processes associated with the atoms of the protein were observed. One at -216mV (pH 8.7) was associated with the Fe-Mo cofactor centres in the protein and allowed identification of the Mössbauer parameters of the oxidized form of these centres. The other redox process at -340mV (pH 8.7) was associated with species M5 [Smith & Lang (1974) Biochem. J. 137, 169-180]. This latter redox process may be involved in enzyme turnover. The oxidized form of species M5 interacts magnetically with species M4. The structural implications of the data have been considered in relation to other published data. It is concluded that an unequivocal assignment of the M4 and M5 atoms to Fe-S cluster types is not yet possible.

scholarly journals The molecular weight of, and evidence for two types of subunits in, the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

1977 ◽  
Vol 163 (3) ◽  
pp. 427-432 ◽  
Author(s):  
R H Swisher ◽  
M L Landt ◽  
F J Reithel
Keyword(s):  
Molecular Weight ◽  
Azotobacter Vinelandii ◽  
Average Molecular Weight ◽  
Amino Acid Content ◽  
Sedimentation Equilibrium ◽  
Weight Average Molecular Weight ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Molecular Weights ◽  
Fe Protein

The weight-average molecular weight of the Mo-Fe protein isolated from Azotobacter vinelandii has been determined by sedimentation-equilibrium techniques. In buffer, the value is 245000+/-5000; in 8M-urea, the value is 61000+/-1000. The protein was separated into two components by chromatography on CM-cellulose in 7M-urea, pH 4.5. These components have similar molecular weights but were shown to differ in charge, amino acid content and arginine-containing peptides. It is proposed that the tetramer has the subunit composition (nalpha2nbeta2).

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