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 Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction

1983 ◽  
Vol 215 (2) ◽  
pp. 393-403 ◽  
Author(s):  
R N F Thorneley ◽  
D J Lowe
Keyword(s):  
Klebsiella Pneumoniae ◽  
Protein Identification ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Substrate Reduction ◽  
Fe Protein ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Kinetics Of

Stopped-flow spectrophotometry and e.p.r. spectroscopy were used to study the kinetics of reduction by dithionite of the oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox.) in the presence of MgADP at 23 degrees C at pH 7.4. The active reductant, SO2.-, produced by the predissociation of S2O4(2-) in equilibrium 2SO2.-, reacts with Kp2ox. (MgADP)2, with k4 = 3.0 X 10(6) +/- 0.4 X 10(6) M-1 X s-1. The inhibition of this reaction by the Mo-Fe protein (Kp1) has enabled the rate of dissociation of Kp2ox. (MgADP)2 from Kp1+ (the Kp2-binding site on Kp1) to be measured (k-3 = 6.4 +/- 0.8 s-1). Comparison with the steady-state rate of substrate reduction shows that the dissociation (k-3) of the complex Kp2ox. (MgADP)2-Kp1+, which is formed after MgATP-induced electron transfer from Kp2 to Kp1+, is the rate-limiting step in the catalytic cycle for substrate reduction.

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 Electron transfer to nitrogenase. Characterization of flavodoxin from Azotobacter chroococcum and comparison of its redox potentials with those of flavodoxins from Azotobacter vinelandii and Klebsiella pneumoniae (nifF-gene product)

1986 ◽  
Vol 239 (1) ◽  
pp. 69-75 ◽  
Author(s):  
J Deistung ◽  
R N F Thorneley
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Gene Product ◽  
Azotobacter Vinelandii ◽  
Azotobacter Chroococcum ◽  
Amino Acid Sequences ◽  
Published Data ◽  
Redox Potentials ◽  
Hydrogen Electrode ◽  
Visible Spectra

Flavodoxin in the hydroquinone state acts as an electron donor to nitrogenase in several nitrogen-fixing organisms. The mid-point potentials for the oxidized-semiquinone and semiquinone-hydroquinone couples of flavodoxins isolated from facultative anaerobe Klebsiella pneumoniae (nifF-gene product, KpFld) and the obligate aerobe Azotobacter chroococcum (AcFld) were determined as a function of pH. The mid-point potentials of the semiquinone-hydroquinone couples of KpFld and AcFld are essentially independent of pH over the range pH 7-9, being -422 mV and -522 mV (normal hydrogen electrode) at pH 7.5 respectively. The mid-point potentials of the quinone-semiquinone couples at pH 7.5 are -200 mV (KpFld) and -133 mV (AcFld) with delta Em/pH of -65 +/- 4 mV (KpFld) and -55 +/- 2 mV (AcFld) over the range pH 7.0-9.5. This indicates that reduction of the quinone is coupled to protonation to yield a neutral semiquinone. The significance of these values with respect to electron transport to nitrogenase is discussed. The amino acid compositions, the N- and C-terminal amino acid sequences and the u.v.-visible spectra of KpFld and AcFld were determined and are compared with published data for flavodoxins isolated from Azotobacter vinelandii.

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).

scholarly journals Electron-paramagnetic-resonance studies on the redox properties of the molybdenum-iron protein of nitrogenase between +50 and −450 mV

1978 ◽  
Vol 173 (3) ◽  
pp. 831-838 ◽  
Author(s):  
M J O'Donnell ◽  
B E Smith
Keyword(s):  
Azotobacter Vinelandii ◽  
Ph Dependence ◽  
Published Data ◽  
Protein Activity ◽  
Active Centre ◽  
Paramagnetic Resonance ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Electron Paramagnetic ◽  
Number Of Bacteria

The midpoint potentials, Em, for the oxidation of the characteristic e.p.r. signal with g values near 4.3, 3.7 and 2.01, of the nitrogenase Mo-Fe proteins from a number of bacteria were measured. They were 0mV for Clostridium pasteurianum, −42mV for Azotobacter chroococcum and Azotobacter vinelandii, −95mV for Bacillus polymyxa and −180mV for Klebsiella pneumoniae Mo-Fe proteins at pH 7.9. The oxidations were thermodynamically reversible for the proteins from A. chroococcum, A. vinelandii and K. pneumoniae and the Em was independent of protein activity for this last protein. The protein from C. pasteurianum required a lower potential for reduction than for oxidation, and the oxidation of the protein from B. polymyxa was only 70% reversible. The apparent Em of the latter protein was decreased by 40mV in the presence of 60mM-MgCl2. The pH-dependence of the Em of the protein from K. pneumoniae was interpreted in terms of a single ionization, not directly associated with the e.p.r.-active centre, with a pKa of 7.0 in the oxidized form of the protein and a pH-independent region at low pH (Em = 118 +/- 6.3 mV). Approx. 20% increase in activity after oxidation was observed for the proteins from B. polymyxa, A. chroococcum and K. pneumoniae. The significance of the above results and their relationship to other published data are discussed.

scholarly journals Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes

1978 ◽  
Vol 173 (1) ◽  
pp. 277-290 ◽  
Author(s):  
D J Lowe ◽  
R R Eady ◽  
R N F Thorneley
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Binding Site ◽  
Dissociation Constants ◽  
Paramagnetic Resonance ◽  
Fe Protein ◽  
Low Concentrations ◽  
Direct Binding ◽  
Electron Paramagnetic ◽  
Single Binding Site

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo–Fe protein showed that they arose from Fe–S clusters in the Mo–Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the −1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme–product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe–S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

scholarly journals The molybdenum–iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ‘mapping’

1976 ◽  
Vol 155 (2) ◽  
pp. 383-389 ◽  
Author(s):  
C Kennedy ◽  
R R. Eady ◽  
E Kondorosi ◽  
D K Rekosh
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl Sulphate ◽  
Azotobacter Vinelandii ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Rhizobium Japonicum ◽  
Fe Protein

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.

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