Short-Term Regulation of the Nitrogenase Activity in Rhodopseudomonas sphaeroides (original) (raw)
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Control of electron transfer in nitrogenase
Current opinion in chemical biology, 2018
The bacterial enzyme nitrogenase achieves the reduction of dinitrogen (N) to ammonia (NH) utilizing electrons, protons, and energy from the hydrolysis of ATP. Building on earlier foundational knowledge, recent studies provide molecular-level details on how the energy of ATP hydrolysis is utilized, the sequencing of multiple electron transfer events, and the nature of energy transduction across this large protein complex. Here, we review the state of knowledge about energy transduction in nitrogenase.
Posttranslational regulation of nitrogenase activity by fixed nitrogen in Azotobacter chroococcum
Biochimica et Biophysica Acta (BBA) - General Subjects, 1996
Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32p]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.
Microbiology, 1986
Nitrogenase activity of suspensions of the unicellular cyanobacterium Gloeothece sp. PCC 6909 plotted against the concentration of dissolved 0, (do,) resulted in a bell-shaped curve, both in the light and in the dark, with optima of 25 or 80 ~M-O , depending on the age of the culture, At the optimum d o 2 , nitrogenase activity [typically 4 to 6 nmol C2H, (mg protein)-' min-l] was similar in the light or in the dark. Alteration of light intensity from zero to 2 klx, or addition of 3-(3,4-dichlorophenyl)-l, 1-dimethylurea (DCMU), had no effect on nitrogenase activity. At 1 klx the ADP/ATP ratio was 0.2 and showed only a marginal increase as the d o 2 was increased. However, a high level of illumination (30 klx) stimulated or inhibited nitrogenase activity, depending on the external do,, presumably as a consequence of changes in the intracellular O2 concentration; in the presence of DCMU, activity increased twofold, independent of do,. In the dark, the dependence of the rate of respiration on O2 concentration suggested the presence of three 0,-uptake systems with apparent K, values of 1 PM, 5 p~ and 25 p~. The ADP/ATP ratio under anaerobic conditions was 0.47 and showed a marked decrease as d o 2 was increased to 25 VM. A CN-insensitive respiratory activity, which neither supported nitrogenase activity nor was coupled to ATP synthesis, was associated with the system with the apparent K, of 5 p~. The dependence of the specific activity of nitrogenase on d o 2 indicated that both the high affinity (K , 1 WM) and low affinity (K , 25 p~) O2-uptake systems contributed ATP or reductant for N,-fixation. KCN (2.5 mM) completely inhibited nitrogenase activity in the dark and at moderate levels of illumination and d o 2. We conclude that respiration is the major source of reductant and ATP for nitrogenase activity both in the dark and in the light, but that photosystem I can contribute ATP at very high levels of illumination.
What is the true nitrogenase reaction? A guided approach
Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology, 2015
Only diazotrophic bacteria, called Rizhobia, living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N2 . In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an extremely high activation energy due to the stable (unreactive) NN triple bond. The structural and functional features of the nitrogenase protein complex, based on the stepwise transfer of eight electrons from reduced ferredoxin to the nitrogenase, coupled to the hydrolysis of 16 ATP molecules, to fix one N2 molecule into two NH3 molecules, is well understood. Yet, a number of different nitrogenase-catalyzed reactions are present in biochemistry textbooks, which might cause misinterpretation. In this article, we show that when trying to balance the reaction catalyzed by the nitrogenase protein complex, it is important to show explicitly the 16 H(+) released by the hyd...
Transinhibition and Voltage-gating in a Fungal Nitrate Transporter
Journal of Membrane Biology, 2003
We have applied enzyme kinetic analysis to electrophysiological steady-state data of Zhou et al. A highaffinity fungal nitrate carrier with two transport mechanisms. J. Biol. Chem. 275:39894-9) and to new current-voltage-time records from Xenopus oocytes with functionally expressed NrtA (crnA) 2H þ -NO À 3 symporter from Emericella (Aspergillus) nidulans. Zhou et al. stressed two Michaelis-Menten (MM) mechanisms to mediate the observed nitrate-induced currents, I NO À 3 . We show that a single straightforward reaction cycle describes the data well, pointing out that during exposure to external substrate, S = (2H + +NO À 3 ) o , the product concentration inside, [P] = ½H þ 2 i Á ½NO À 3 i , may rise substantially near the plasma membrane, violating the condition [P] ( [S] for MM kinetics. Here, [P] and its changes during experimentation are treated explicitly. K 1/2 » 20 lM for I NO À 3 at pH o from Zhou et al. is confirmed.
Energy Transduction in Nitrogenase
Accounts of chemical research, 2018
Nitrogenase is a complicated two-component enzyme system that uses ATP binding and hydrolysis energy to achieve one of the most difficult chemical reactions in nature, the reduction of N to NH. One component of the Mo-based nitrogenase system, Fe protein, delivers electrons one at a time to the second component, the catalytic MoFe protein. This process occurs through a series of synchronized events collectively called the "Fe protein cycle". Elucidating details of the events associated with this cycle has constituted an important challenge in understanding the nitrogenase mechanism. Electron delivery is a multistep process involving three metal clusters with intra- and interprotein events. It is proposed that the first electron transfer event is a gated intraprotein transfer of one electron from the MoFe protein P-cluster to the FeMo cofactor. Measurement of the effect of osmotic pressure on the rate of this electron transfer process revealed that it is gated by protein co...