Lecture 22 PMB

Pregunta	Respuesta
Limiting nutrients for plant growth and productivity	Nitrogen, water, carbon
Considering nitrogen is so abundant in the atmosphere, why is it a limiting factor in plant growth?	N2 is very stable but must be fixed/reduced to be used by plants
basic reaction of Nitrogen fixation	N2 reduced to NH4+ (NH3), which can then be converted to biomolecules
Haber Bosch industrial process	Nitrogen and hydrogen reacted over iron at 350C and >200atm N2(g) + 3H2(g) ↔ 2NH3(g) fertilizer from this supports 1/3 of our population!
Steps in nitrogen cycle performed exclusively by bacteria and archaea	Nitrogen fixation: N2 --> NH3 Nitrification: NH3 --> NO3-
Steps in nitrogen cycle that are not necessarily performed by bacteria or archaea	Denitrification: NO3- --> N2 Ammonification/Assimilation & Assimilation: NH3 --> NH2 groups, organic compounds and proteins --> NO3-
chemical fertilizer PROS	INCREASED CROP YIELD
chemical fertilizer CONS	fertilizer CONTAMINATES ground water and consumes fossil fuel resources!!
Chemical vs Biological N fixation	N2 triple bond is hard to break, even though ΔG is negative a lot of energy (high Temp, Pressure, and iron catalyst) is required for reaction biological fixation occurs at low Temp, 1 atm, with a nitrogenase enzyme as the catalyst; energy supplied from ATP
Equation for biological nitrogen fixation	N2 + 8H+ + 8e- + 16MgATP --> 2NH3 + 16MgADP + 16Pi + H2 e- come from reduced Fd or flavodoxin these numbers are all a minimum!! 16-24 ATP typically used
Options for nitrogenase reduction reaction	Nitrogenase can reduce N2 to ammonia or acetylene to ethylene: 1) N2 + 8H+ + 8e- --> 2NH3 + H2 2) C2H2 + 2H+ + 2e- --> C2H4 both break a triple bond
How to demonstrate nitrogenase activity	digest cells containing stable isotope 15N2 --> they will release NH3 from biomolecules --> shown to contain 15N by mass spec OR (easier) reduce acetylene to ethylene with analysis in a gas chromatograph shows production of C2H4 from C2H2 over time
How do you get DEFINITIVE proof of N2 fixation	grow cells in sealed containers, give air at top if aerobic or regular N2 gas/etc for anaerobic → inject nitrogenase → after periods of time withdraw samples from gas and see if acetylene (C2H2) has been converted to ethylene (C2H4)
proteins within nitrogenase complex	dinitrogenase reductase = Fe protein, accepts e- from Fdred and binds ATP --> passes e- to FeMo and hydrolyzes ATP dinitrogenase = FeMo protein, uses e- to reduce bound N2 to NH3 both rxn centers VERY susceptible to O2
How many ATPs are hydrolyzed when e- move from Fe to FeMo cofactor?	4 ATP hydrolyzed per step 4 steps total, so 24 ATP used up
Nitrogenase complex reaction cycle	1) Fe protein gets 2e- from an e-donor (Fdox) and binds 4 ATP 2) Transfers 2 e- to FeMo protein and hydrolyzes 4 ATP 3) FeMo protein binds 2H+ --> reduced by the e- to H2 4) N2 displaces H2 on the FeMo protein 5) Fe protein reduced by Fd(red) 3X more times, total of 8e- and 16 ATP 6) at each turn of 5, 2e- transferred to bound N2, yielding 2NH3 7) NH3 is found as the soluble ion NH4+
How do phototrophic organisms reduce ferrodoxin? (considering Fdred is a strong donor)	Cyanobacteria use light energy and PSI to create Fd(red) review end of Z-scheme
How do non-phototrophic ANAEROBES organisms reduce ferrodoxin? (considering Fdred is a strong donor)	use pyruvate-ferrodoxin oxidoreductase Pyruvate + CoA + Fd(ox) → Acetyl-CoA + CO2 + Fd(red)
How do non-phototrophic AEROBES organisms reduce ferrodoxin? (considering Fdred is a strong donor)	use reverse electron transfer from NADH/anything with higher reduction potential than Fd Energy in the form of ATP or PMF is used! --> may account for the fact that nitrogen fixation uses more than the predicted 16 ATP per 2NH3
How do cyanobacteria protect nitrogenase from O2, considering their phototrophy produces O2?	1) Temporal separation of photosynthesis and nitrogen fixation 2) Spatial separation of the two processes: vegetative cells for photosynthesis and heterocysts for nitrogen fixation
heterocysts	terminally differentiated cell in cyanobacteria where photosystem II is dismantled and oxidases are produced to keep the O2 concentration very low
How do obligate aerobes protect nitrogenase from O2?	A. vinelandii : obligate aerobe soil bacteria 1) produces slime layer --> limits O2 influx in cell 2) VERY high respiratory rate --> removes O2 quickly 3) protective protein binds to nitrogenase and protects it from damage by O2 most important of the three
How do symbionts protect nitrogenase from O2?	S. meliloti: aerobic gram negative symbiont of alfalfa grass --> form root nodules that develop into bacteroids with N2-fixing ability --> in nodule, leghemoglobin produced by the plant binds O2 and reduces free [O2] free-living S. meliloti does not have genes required for N2 fixation in presence of O2
nif regulon	genes required for N2 fixation! ONLY expressed when needed (no NH4+) & when they could function properly (low O2) NifA is positive regulator; control of expression (via NtrC) and activity (via NifL) important for appropriate response to environmental conditions
N2 fixation regulation in FREE-LIVING Klebsiella pneumoniae	Presence of O2 OR NH4+ blocks nif tx!! NtrB: His kinase activated by LOW [NH4+] NtrC: DNA-binding response regulator σ54: positive regulator of N-scavenging & N2-fixing genes, works with NtrC NifA: positive regulator of nif genes NifL: negative regulator of NifA, binds to NifA in presence of O2 to block nif tx
how is NtrB kinase activity promoted?	cells are effectively looking at their C/N ratio REVIEW THIS
Regulation of nitrogen fixation genes in S. meliloti during symbiosis	O2 blocks FixL --> blocks nif and fix tx FixL: His kinase with heme sensing domain FixJ: DNA-binding response regulator NifA and FixK: tx factors activate expression of nif and fix genes Other nif and fix genes also involved!

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