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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
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nitrogen fixation
- conversion of atmosphere N2 to NH3
- critical to all life - NH4+ is converted into biochemicals
- only done by bacteria and archae
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the Haber-Bosch process
- nitrogen and hydrogen are reacted over an iron catalyst over conditions of 200 atm, 450° C
- used to make fertilizer
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pros and cons of commercial fertilizers
- increased crop yield
- contaminates the groundwater, consumes fossil-fuel resources
- biological nitrogen fixation is an alternative
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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
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the nitrogen fixation reaction
- N2+3H2 <-> 2NH3thermodynamically favorable
- triple bond of N2 is very hard to break - a lot of energy required for this reaction
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use of 15N to prove biological N2 fixation, Robert Burris
- stable isotope
- can trace using mass spec to tell difference between 14N and 15N
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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
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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
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cyanobacteria and limiting O2 exposure of nitrogenase
- temporal separation:
- photosynthesis - day
- nitrogen fixation - night
- spatial separation:
- heterocysts - specialized cells do nitrogen fixation
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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
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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
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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
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