Bio 119 Nitrogen Metabolism 07/08/09

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Lawrence Mitchell
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1 Nitrogen Fixation READING: BOM Nitrogenase and Nitrogen Fixation p. 605 Nitrogenase Electron Flow in Nitrogen Fixation Alternative Nitrogenases The Unique Nitrogenase of Streptomyces thermoautotrophicus Assaying Nitrogenase: Acetylene Reduction Genetics and Regulation of N 2 Fixation p. 608 Genetics of Nitrogen Fixation Regulation of Nitrogenase Synthesis Regulation of Nitrogenase Activity 15.9 Free-Living Aerobic Nitrogen-Fixing Bacteria p. 416 Taxonomy Azotobacter and Alternative Nitrogenases Cyanobacteria p. 463 We are covering ONLY the information on heterocysts in Sec (including Fig ) on p The Legume-Root Nodule Symbiosis p. 725 Leghemoglobin and Cross-Inoculation Groups Steps in Root Nodule formation Attachment and Infection Bacteroids Nodule Formation: Nod Genes, Nod Proteins, and Nod factors Biochemistry of Root Nodules Stem-Nodulating Rhizobia Nonlegume Nitrogen-Fixing Symbioses: Azolla-Anabaena and Frankia 1 of 7

2 20.14 Nitrogenase and Nitrogen Fixation p. 605 Global N 2 Fixation: Lightning, other non-biological 10 Tg/yr Biological 50 Industrial (Haber-Bosch) 110 TOTAL 170 Tg/yr Tg = g N H 2 2 NH 3 ΔG ' = kj/mole Although the reduction of molecular nitrogen to ammonia is exergonic, the triple bond is very stable, and the activation energy is a whopping 930 kj/mole. The Haber process achieves the activation energy is achieved by raising temperature to 500 C and pressure to several hundred atmospheres. This is the basis for the fossil fuel dependence of synthetic nitrogen fertilizer production. By some accounts, 2% - 5% of all fossil fuel consumption is allocated to industrial N 2 fixation. BIOLOGICAL DI-NITROGEN FIXATION OVERALL REACTION N N [HN NH] [ H 2 N NH 2 ] NH 3 + NH 3 H 2 Nitrogenase Complex hydrolyses 2 ATP for each of the 8 electrons involved with the overall reaction. i.e. 16 ATP per N 2. Nitrogenase Complex is: Widely but sporadically distributed in Bacteria and Archaea, probably as a reslt of lateral gene transfer; absent in Eukarya except via symbiosis Highly conserved in structure and function. Very sensitive to inhibition by O 2. Uses 2 unusual redox active prosthetic groups (P-Cluster and Fe Mo Cofactor) 2 of 7

3 Structure of the Nitrogenase Complex Dinitrogenase Dinitrogenase Reductase Fe S ATP P Cluster (2 4Fe 4S) Fe Mo Co The Nitrogenase Complex consists of: DINITROGENASE = 240kD (2 X 2 subunits); P Cluster; Fe Mo Cofactor N2 binding to Fe Mo Cofactor (?) DINITROGENASE REDUCTASE = 60 kd (2 X 1 subunit); 1 X 4Fe 4S center; 2 ATP binding sites P Clusters are "bridged" 4Fe 4S centers 3 of 7

Each Reductase subunit delivers only 1 e- to dinitrogenase. The oxidized reductase is released from the complex to be reduced by Ferredoxin (or other strong electron donator). -0.

608 Genetics of Nitrogen Fixation This description applies to the enteric bacterium Klebsiella. Klebsiella is a Gammaproteobacterium closely related to E. coli.

4 Each Reductase subunit delivers only 1 e- to dinitrogenase. The oxidized reductase is released from the complex to be reduced by Ferredoxin (or other strong electron donator) V FERREDOXIN REDUCTASE 2 ADP DINITROGENASE ε o ' -0.3 V REDUCTASE 2 ATP N V Genetics and Regulation of N 2 Fixation p. 608 Genetics of Nitrogen Fixation This description applies to the enteric bacterium Klebsiella. Klebsiella is a Gammaproteobacterium closely related to E. coli. It is a facultative anaerobe, and fixes nitrogen only while growing under anoxic conditions. Note that the 7 operons in the nif regulon are contiguous. There is no functional reason for the the operons to be contiguous, but this facilitates lateral gene transfer. Also note that while more than half the genes in the regulaon are involved with FeMoCo metabolism, there are no genes for the P Cluster. nitrogenase structural genes (3) nifd dinitrogenase α nifk dinitrogenase β nifh dinitrogenase reductase α 4 of 7

5 Fe Mo Co biosynthesis (12) Mo processing (1) electron transport (2) unknown (1) nifn, V, Z, W, E, B, S, U, X, N, E, Y (Fe Mo Co insertion) nifq niff flavodoxin nifj pyruvate flavodoxin oxidoreductase nift Regulation of Nitrogenase Synthesis regulation (2) nifa positive regulation of nif transcription by low NH3 (via activation by NtrC) nifl negative regulation by O 2 (FAD) Regulation of Nitrogenase Activity Proteobacteria: ammonia switch-off - covalent modification of DR in response to elevated NH3 levels Archaea/Clostridium: Inhibitory protein binds to nitrogenase in response to lowered α-ketoglutarate levels 15.9 Free-Living Aerobic Nitrogen-Fixing Bacteria p. 416 Taxonomy Free-Living Symbiotic Gammaproteobacteria Azotobacter Klebsiella Azomonas Alphaproteobacteria Azospirillum Rhizobium Beijerinckia Betaproteobacteria Derxia Gram-Positive Clostridium Frankia Cyanobacteria Anabaena Anabaena (with Azolla) Azotobacter and Alternative Nitrogenases "backup" nitrogenases use FeV or Fe Cyanobacteria p. 463 Structural Variations: Gas Vesicles and Heterocysts This is an example of multicellualrity with cell differentiation and coordination. We are not covering gas vesicles here. 5 of 7

6 24.15 The Legume-Root Nodule Symbiosis p. 725 All the "rhizobia" are members of the Alphaproteobacteria. Leghemoglobin and Cross-Inoculation Groups Rhizobia fix nitrogen under microaerophilic conditions in culture. Microaerophilic conditions are maintained in root nodules by leghemoglobin. (Plant makes protein; rhizobis make heme group.) Steps in Root Nodule formation Attachment and Infection Recognition: Plant roots secrete diverse organic "stimulants" Proliferation of rhizobia in rhizosphere IG-specific attachemnt to root hairs Attachment involves plant membrane protein rhicahesin and glycoprotein lectins Nod factor-induced root hair curling Root cell penetration Infection-thread cellulosic tube formation Infection spreads to adjacent cells and plant cell proliferation produces nodules. Bacteroids Rhizobia multiply and terminally differentiate into bacteroids in symbiosome; no longer capable of growth in culture. Nif gene expression. Nodule Formation: Nod Genes, Nod Proteins, and Nod factors Nod Genes (10) nodabc: nod? nodd: common oligosaccharide nod factor production IG-specific modification pf nod oligosaccharide backbone positive nod gene transcription regulation in response to plant flavonoids (IG specificity) Biochemistry of Root Nodules Stem-Nodulating Rhizobia Nonlegume Nitrogen-Fixing Symbioses: Azolla-Anabaena and Frankia/Alnus 6 of 7

7 DISCUSSION QUESTIONS: 1. How is nitrogenase protected from O2 inactivation in Rhizobium, in Azotobacter, Frankia and in Anabaena? 2. Nitrogen fixation in Klebsiella requires the expression of about 20 genes in the nif regulon. Provide a list of the functions of the 20 gene products. 3. What differences have been observed between the ammonia switch-off effect in Proteobacteria and Archaea? 4. Which metals substitute for Mo in the "alternative" nirogenases in Azotobacter chroococcum? 4. What arguments can be produced to support the claim that N2 fixation is an ancient, highly conserved process? 5. Describe the general mechanism of the nitrogenase reaction. 7 of 7

Nitrogen fixation and cycle

Nitrogen fixation and cycle . Paper : 04 Module : 17 Principal Investigator, Paper Coordinator and Content Writer Dr. Sunil Kumar Khare,Professor Dept. of Chemistry, I.I.T. Delhi Content Reviewer: Prof. Prashant Mishra, Professor