Nitrogen Fixation

  1. necessity of nitrogen fixation
    • nitrogen is a major limiting nutrient even though nitrogen gas is abundant
    • N2 is chemically very stable - must be fixed before it is assimilated by living organisms
  2. nitrogen fixation
    • conversion of atmosphere N2 to NH3
    • critical to all life - NH4+ is converted into biochemicals
    • only done by bacteria and archae
  3. the Haber-Bosch process
    • nitrogen and hydrogen are reacted over an iron catalyst over conditions of 200 atm, 450° C
    • used to make fertilizer
  4. pros and cons of commercial fertilizers
    • increased crop yield
    • contaminates the groundwater, consumes fossil-fuel resources
    • biological nitrogen fixation is an alternative
  5. bacterial N2 fixation
    • only some prokaryotes perform
    • nodule-associated - symbiosis between Rhizobium and leguminous plants, agricultural importance
    • free-living nitrogen fixers, no direct agricultural importance, ex-Azobacter has slime and high respiratory rate
  6. the nitrogen fixation reaction
    • N2+3H2 <-> 2NH3
    • thermodynamically favorable
    • triple bond of N2 is very hard to break - a lot of energy required for this reaction
  7. use of 15N to prove biological N2 fixation, Robert Burris
    • stable isotope
    • can trace using mass spec to tell difference between 14N and 15N
  8. the acetylene reduction assay
    use ability of nitrogenase to catalyze formation of ethylene from acetylene to assay for nitrogenase activity

    • have stoppered vial containing cell suspension and atomospheric air
    • incubate, if nitrogenase present get acetylene -> ethylene conversion
    • sample headspace periodically and inject into gas chromatograph
    • results show no ethylene when experiment begins, but increasing ethylene production as acetylene is consumed
  9. nitrogenase enzyme complex
    • Dinitrogenase reductase:
    • Fe protein
    • 4Fe-4S cluster
    • homodimer

    • dinitrogenase:
    • FeMo protein
    • P-cluster (Fe-S cluster)
    • FeMo-co

    • electrons are transferred from DNR to dinitrogenase:
    • electrons are passed from 4Fe-4S cluster to P-cluster to FeMo cluster

    • the binding of dinitrogenase reductase to dinitrogenase requires binding of ATP to DNR - alters the conformation of DNR and allows it to bind dinitrogen
    • pyruvate to flavoprotein or ferredoxin can be electron donor
  10. cyanobacteria and limiting O2 exposure of nitrogenase
    • temporal separation:
    • photosynthesis - day
    • nitrogen fixation - night

    • spatial separation:
    • heterocysts - specialized cells do nitrogen fixation
  11. How is nitrogenase protected from O2 in Azotobacter vinelandii?
    • produces a slime, limits O2 in cell
    • very high respiratory rate
    • protective protein that binds to nitrogenase, protecting it against irreversible inhibition by O2
  12. How is nitrogenase protected from O2 in Sinorhizobium meloti symbiont of alfalfa?
    • Leghemoglobin produced by plant in nodule binds O2, dramatically reducing O2 exposure of nitrogenase
    • Do not get expression of genes required for N2 fixation in presence of O2
  13. transcriptional regulation of nitrogen fixation*
    • many enzymes required, must be coordinately expressed but costly to express them
    • two component system induces genes only in low oxygen environment or when needed - no NH4+

    • NtrB-NtrC = two component NH4+ dependent regulation
    • NtrC is phosphorylated and activates transcription of NifLA regulators when NH4+ is low
    • NtrB is the sensor kinase that is responsive to N status
    • NifA is the postive regulator of nitrogenase gene expression, only active when O2 is very limited
    • In the presence of O2, NifL binds to NifA preventing NifA from binding to its binding site and activating expression of nitrogenase genes
Card Set
Nitrogen Fixation
general microbiology midterm 2