Kinetics of nitrogenase of <i>Klebsiella pneumoniae</i>. Heterotropic interactions between magnesium-adenosine 5′-diphosphate and magnesium-adenosine 5′-triphosphate

Thorneley, R. N. F.; Cornish‐Bowden, Athel

doi:10.1042/bj1650255

Cited by 40 publications

(40 citation statements)

References 33 publications

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“…Our conclusion is that during nitrogenase activity, the ATP/ADP ratio is less than 1 and increases to 3 to 4 when nitrogenase is O 2 inhibited. Studies with isolated enzyme indicate that MgADP is a strong inhibitor of nitrogenase (30). It is questionable whether nitrogenase activity is possible at the low ATP/ADP ratios found.…”

Section: Discussionmentioning

confidence: 99%

“…Aside from an anaerobic environment and a source of reducing equivalents, MgATP is necessary in vitro for nitrogenase activity. MgADP inhibits nitrogenase (30). This introduces a major problem inherent to aerobic nitrogen-fixing organisms, namely that O 2 is essential for ATP synthesis and probably also for electron transport to nitrogenase (14), and on the other hand, O 2 inhibits and inactivates nitrogenase.…”

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confidence: 99%

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Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids

et al. 1996

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The relationship between the O 2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O 2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O 2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 M O 2 , a decline in the O 2 consumption rate was observed to 50 to 70% of the maximal O 2 consumption rate. A determination of the redox state of the cytochromes during an O 2 consumption experiment indicated that at O 2 concentrations above 5 M, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O 2 concentrations of >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (K m of 45 ؎ 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O 2 .Nitrogen fixation is essentially an anaerobic process. The enzyme nitrogenase, which catalyzes the reduction of N 2 to NH 3 , consists of two O 2 -labile proteins (16,25). The physiological electron donors for nitrogenase, flavodoxin and ferredoxin, are auto-oxidizable (35). Aside from an anaerobic environment and a source of reducing equivalents, MgATP is necessary in vitro for nitrogenase activity. MgADP inhibits nitrogenase (30). This introduces a major problem inherent to aerobic nitrogen-fixing organisms, namely that O 2 is essential for ATP synthesis and probably also for electron transport to nitrogenase (14), and on the other hand, O 2 inhibits and inactivates nitrogenase. This paradox has been resolved in the symbiosis of Rhizobium or Bradyrhizobium species with legumes by the organogenesis of the root nodule. In the central zone of the root nodule a microaerobic environment is created. The microaerobic condition is maintained by a regulation of the influx of O 2 into the central tissue (21), which balances the O 2 uptake of the mitochondria and the bacteroids. Furthermore, a...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids

et al. 1996

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“…(Smith et al, , 1973, Mossbauer (Smith & Lang, 1974) and stopped-flow (Thorneley, 1975;Thorneley & Cornish-Bowden, 1977) spectroscopy showed that Kp2 protein donates electrons to Kpl protein in a MgATP-dependent reaction that is rapid (k = 2.0x 102s-' at 23°C) relative to the catalytic-centre activity for the enzyme (approx. 2s-1).…”

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confidence: 99%

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

Lowe¹,

Eady²,

Thorneley³

1978

Biochemical Journal

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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.

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“…MgADP complex might explain the strong inhibition of the nitrogenase reaction by MgADP. The absence of the second electron prevents catalysis rather than competition with bound MgATP as proposed [36]. APPENDIX Scheme 1 gives the basic steps, as a minimal hypothesis, necessary to explain the phenomena observed.…”

Section: Xymentioning

confidence: 99%

The Effect of the Redox Potential on the Activity of the Nitrogenase and on the Fe-Protein of Azotobacter vinelandi

et al. 1982

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The redox potential dependence on the nitrogenase reaction catalyzed by different nitrogenase cornplexcs from Azotohacter vinelundii has been studied by two methods. The redox potential was set with the redox couplc SO;p/SO:-and the effect on the nitrogenase activity was determined. The oxidation of photochemically reduced low-potential electron carriers by nitrogenase was followed spectrophotometrically. Simultaneously the nitrogenase activity was estimated by the production of hydrogen. It was found that when component I1 of the nitrogenase is in redox equilibrium with the electron donor, the nitrogenase activity is maximum and independent of the redox potential up to -440 mV. At higher potentials the nitrogenase activity declines and no significant activity was detectable at potentials above -350 mV.The effect of the redox potential on the oxidation reduction state of the 4Fe-4S cluster of Azotohacter nitrogenase component I1 was studied by electron paramagnetic resonance spectroscopy. Without adenine nucleotides present, component I1 of the nitrogenase shows a midpoint potential of -393 mV and the oxidation reduction reaction is characterized by the transfer of two electrons per redox step. In the presence of MgATP the midpoint potential of component I1 of the nitrogenase undergoes a negative shift of 42 mV. The curve fitting to the experimental points is also characteristic of a two-electron redox step. In contrast, it is found that in the presence of MgADP, the curve fitting to the experimental points is characterized by a one-electron step with a midpoint potential of -473 mV. It is demonstrated that the nitrogenase activity correlates with the oxidation reduction state of component I1 of the nitrogenase without adenine nucleotides bound. The implications of this observation, indicating a sequential reaction of the reduced Fe protein with the Mo-Fe protein, for thc present kinetic models of nitrogenase will be discussed.

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

Cited by 40 publications

References 33 publications

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids

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

The Effect of the Redox Potential on the Activity of the Nitrogenase and on the Fe-Protein of Azotobacter vinelandi

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