Nitrogenase of Azotobacter chroococcum Kinetics of the reduction of oxidized iron–protein by sodium dithionite

1976 ◽  
Vol 157 (3) ◽  
pp. 529.b1-529.b1
Author(s):  
R N F Thorneley ◽  
M G Yates ◽  
D J Lowe
Keyword(s):  
Sodium Dithionite ◽  
Azotobacter Chroococcum ◽  
Iron Protein ◽  
Oxidized Iron ◽  
Kinetics Of

scholarly journals Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite

1976 ◽  
Vol 155 (1) ◽  
pp. 137-144 ◽  
Author(s):  
R N F Thorneley ◽  
M G Yates ◽  
D J Lowe
Keyword(s):  
Electron Paramagnetic Resonance Spectroscopy ◽  
Sodium Dithionite ◽  
Azotobacter Chroococcum ◽  
High Rate ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Fe Protein ◽  
Catalytically Active ◽  
Electron Reduction ◽  
Kinetics Of

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2- with k greater than 10(8)M-.S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2- at 23 degrees C. Further reductions by SO2- occurred in three slower phases with rate constants in the range 10(4) −10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-of the Fe-protein alone (k greater than 108M1.S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 } 102s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.

scholarly journals The vanadium- and molybdenum-containing nitrogenases of Azotobacter chroococcum. Comparison of mid-point potentials and kinetics of reduction by sodium dithionite of the iron proteins with bound magnesium adenosine 5′-diphosphate

1988 ◽  
Vol 251 (1) ◽  
pp. 165-169 ◽  
Author(s):  
J Bergström ◽  
R R Eady ◽  
R N F Thorneley
Keyword(s):  
Sodium Dithionite ◽  
Azotobacter Chroococcum ◽  
Effective Electron ◽  
Iron Proteins ◽  
Fe Protein ◽  
Protein Components ◽  
Dithionite Ion ◽  
Kinetics Of ◽  
Kinetics Of Reduction

The mid-point potentials of the Fe protein components (Ac2 and Ac2* respectively) of the Mo nitrogenase and V nitrogenase from Azotobacter chroococcum were determined in the presence of MgADP to be −450 mV (NHE) [Ac2(MgADP)2-Ac2*ox.(MgADP)2 couple] and −463 mV (NHE) [Ac2* (MgADP)2-Ac2*ox.(ADP)2 couple] at 23 degrees C at pH 7.2. These values are consistent with a flavodoxin characterized by Deistung & Thorneley [(1986) Biochem. J. 239, 69-75] with Em = −522 mV (NHE) being an effective electron donor to both the Mo nitrogenase and the V nitrogenase in vivo. Ac2*ox.(MgADP)2 and Ac2*ox.(MgADP)2 were reduced by SO2.- (formed by the predissociation of dithionite ion, S2O4(2-)) at similar rates, k = 4.7 × 10(6) +/- 0.5 × 10(6) M-1.s-1 and 3.2 × 10(6) +/- 0.2 × 10(6) M-1.s-1 respectively, indicating structural homology at the electron-transfer site associated with the [4Fe-4S] centre in these proteins.

scholarly journals Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments

1989 ◽  
Vol 261 (1) ◽  
pp. 181-187 ◽  
Author(s):  
R N F Thorneley ◽  
G A Ashby
Keyword(s):  
Azotobacter Chroococcum ◽  
Stopped Flow ◽  
Protein Species ◽  
Respiratory Pathway ◽  
Kinetics Of Oxidation ◽  
Iron Protein ◽  
Respiration Rates ◽  
High Respiration Rate ◽  
Nitrogenase Iron Protein ◽  
Kinetics Of

The kinetics of oxidation of the Fe proteins of nitrogenases from Klebsiella pneumoniae (Kp2) and Azotobacter chroococcum (Ac2) by O2 and H2O2 have been studied by stopped-flow spectrophotometry at 23 degrees C, pH 7.4. With excess O2, one-electron oxidation of Kp2 and Ac2 and their 2 MgATP or 2 MgADP bound forms occurs with rate constants (k) in the range 5.3 x 10(3) M-1.S-1 to 1.6 x 10(5) M-1.S-1. A linear correlation between log k and the mid-point potentials (Em) of these protein species indicates that the higher rates of electron transfer from the Ac2 species are due to the differences in Em of the 4Fe-4S cluster. The reaction of Ac2(MgADP)2 with O2 is sufficiently rapid for it to contribute significantly to the high respiration rate of Azotobacter under N2-fixing conditions and may represent a new respiratory pathway. Excess O2 rapidly inactivates Ac2(MgADP)2 and Kp2(MgADP)2; however, when these protein species are in greater than 4-fold molar excess over the concentration of O2, 4 equivalents of protein are oxidized with no loss of activity. The kinetics of this reaction suggest that H2O2 is an intermediate in the reduction of O2 to 2 H2O by nitrogenase Fe proteins and imply a role for catalase or peroxidase in the mechanism of protection of nitrogenase from O2-induced inactivation.

Kinetics of anthraquinone reduction with sodium dithionite in alkaline medium

AIChE Journal ◽  
1988 ◽  
Vol 34 (5) ◽  
pp. 865-869 ◽  
Author(s):  
J. C. Burillo ◽  
Francisco Rodríguez ◽  
L. F. Adrados ◽  
J. F. Tijero
Keyword(s):  
Alkaline Medium ◽  
Sodium Dithionite ◽  
Kinetics Of

Kinetics of oxidation of sodium dithionite by flow thermal method

AIChE Journal ◽  
1978 ◽  
Vol 24 (2) ◽  
pp. 232-237 ◽  
Author(s):  
D. K. Singh ◽  
R. N. Sharma ◽  
R. D. Srivastava
Keyword(s):  
Sodium Dithionite ◽  
Thermal Method ◽  
Kinetics Of Oxidation ◽  
Kinetics Of

Effect of the temperature on the kinetics of the chemisorption of atmospheric oxygen by a sodium dithionite solution

2006 ◽  
Vol 80 (9) ◽  
pp. 1481-1483
Author(s):  
S. V. Aleeva ◽  
G. V. Chistyakova ◽  
S. A. Koksharov ◽  
Yu. V. Polenov
Keyword(s):  
Atmospheric Oxygen ◽  
Sodium Dithionite ◽  
Kinetics Of

scholarly journals Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre

1987 ◽  
Vol 246 (2) ◽  
pp. 455-465 ◽  
Author(s):  
G A Ashby ◽  
R N F Thorneley
Keyword(s):  
Klebsiella Pneumoniae ◽  
Rate Constants ◽  
Protein Conformation ◽  
Kinetic Studies ◽  
Sodium Dithionite ◽  
Conformation Change ◽  
Fe Protein ◽  
Redox Active ◽  
Dithionite Ion ◽  
Kinetics Of

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 × 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 × 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 × 10(5) less than k less than 3 × 10(6) M-1.s-1 and protein conformation change 6 × 10(2) less than k less than 6 × 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.

scholarly journals The binding of cyanide to ferroperoxidase

1971 ◽  
Vol 122 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Charles Phelps ◽  
Eraldo Antonini ◽  
Maurizio Brunori
Keyword(s):  
Sodium Dithionite ◽  
Affinity Constant ◽  
Kinetic Measurements ◽  
First Order ◽  
Ph Values ◽  
Cyanide Binding ◽  
Single Process ◽  
Ph Jump ◽  
Equilibrium And Kinetics ◽  
Kinetics Of

1. The equilibrium and kinetics of cyanide binding to ferroperoxidase were investigated. At pH9.1 the equilibrium and kinetic measurements agree closely and disclose a single process with an affinity constant of 1.1×103m@!-1 and combination and dissociation velocity constants of 29m-1·s-1 and 2.5×10-2s-1 respectively. 2. At pH values below 8 the affinity constant falls until at pH6.0 the ferroperoxidase·cyanide complex is no longer formed. This is shown to be associated with the formation of ferriperoxidase·cyanide complex in the mixture even in the presence of excess of sodium dithionite. 3. Rapid-pH-jump experiments show a fast pseudo-first-order interconversion between ferroperoxidase·cyanide complex at pH9.1 and ferriperoxidase·cyanide complex at pH6.0. 4. The kinetics of binding of cyanide to dithionite-reduced peroxidase at pH6.0 are complicated and radically different from those observed at pH9.1. 5. Above pH8 the change of affinity constant with pH is consistent with the undissociated species, HCN, being bound by the ferroperoxidase. The enthalpy for this process measured both by equilibrium and kinetic methods is about -8kcal/mol. 6. The binding of cyanide to reconstituted peroxidases, proto, meso and deutero, was investigated. 7. The results are discussed in relation to known data on cyanide binding to other haemoproteins.

Kinetics of the Reduction of p-Phenylazobenzenesulphonic Acid by Sodium Dithionite in Alkaline Solution

2008 ◽  
Vol 81 (9) ◽  
pp. 403-405 ◽  
Author(s):  
C. R. WASMUTH ◽  
R. L. DONNELL ◽  
C. E. HARDING ◽  
G. E. SHANKLE
Keyword(s):  
Alkaline Solution ◽  
Sodium Dithionite ◽  
Kinetics Of
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