Download Lec 15: Nitrogen in biochemistry

Lec 15: Nitrogen in biochemistry
Reference material
Biochemistry 4th edition, Mathews, Van Holde, Appling, Anthony‐Cahill. Pearson ISBN:978‐0‐13‐800464‐4
Lehninger Principles of Biochemistry 4th edition, David L. Nelson, Michael M. Cox. W. H. Freeman ISBN:978‐0716743392
Nitrogen metabolism
Third 1/3 of class
國立交通大學生物科技學系 蘭宜錚老師
First 1/3 of class
second 1/3 of class
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Why is nitrogen important in biology?
2 of the 4 major biomolecules require Nitrogen:
Amino acids
Nucleic acids Proteins
DNA & RNA
Enzymes are needed to catalyze reactions of life
DNA and RNA are needed for information relay
Nitrogen availability limits growth and reproduction of many organisms
When nitrogen (and other nutrients) is leaked into environment..
Leaks of Nitrogen, Phosphorus, and other organics lead to undesirable algal blooms.. which is toxic
國立交通大學生物科技學系 蘭宜錚老師
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Biological Nitrogen
Different forms of Nitrogen can be utilized by life.. But of course with preference
Require a lot of energy! & Directly useable for making cellular Anaerobic only!
Most important/central
machinery such as proteins and DNA
Nitrogen Ammonia Fixation
N2
assimilation
Proteins
Amino acids Biosynthesis
NH3
Nitrogen
DNA
ammonia
Nucleic acids
gas
Denitrification
ammonification (small molecules Degradation (complex Nitrogen that contain nitrogen, “Organic nitrogen”)
NO2‐
Nitrite
Require energy input!
NO3‐
Nitrate
containing molecules)
Priority (or ease of utilization):
1. Directly use proteins, amino acids, etc.
2. Use other organic Nitrogen containing
small molecules (“organic nitrogens”)
3. Ammonia
4. Nitrate and nitrite
5. Nitrogen gas (N2) fixation
Biological Nitrogen availability
Most abundant source of Nitrogen is N2 gas
Earth’s atmosphere is made up of:
78% Nitrogen (N2)
21% Oxygen (O2)
0.9% Argon (Ar)
0.25% H2O *average*
0.04% CO2
Although N2 is abundant in Earth environment, its direct utilization is limited to few organisms (for example Rhizobium living around roots of legume plants). The organisms that fix N2 are called Diazatrophs
Therefore most other organisms rely on these N2‐
fixing microbes to generate NH3
Roots
Legume root nodules
For life to thrive, N2 fixation to NH3 is very important!
國立交通大學生物科技學系 蘭宜錚老師
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Nitrogen fixation by Nitrogenase
• While the formation of NH3 from N2 and H2 is a favorable reaction, the activation
energy is extremely high as the N N bond is very stable with bond energy of 930 kJ/mol
• Naturally, N2 is fixed using an enzyme called Nitrogenase, which catalyzes the
following reaction:
16 ATP are used to drive this reaction
• 6 e‐ go to N2, forming 2 NH3.
• 2 e‐ go and form H2 as a byproduct
Nitrogenase is oxygen sensitive
• Nitrogenases are oxygen‐sensitive, which becomes irreversibly deactived upon
exposure to oxygen.
• Different organisms have developed different methods to overcome oxygen‐
sensitivity.
• Formation of heterocysts in diazatrophic cyanobacteria
• Production of additional cell walls
• Glycolipid to form hydrophobic barrier
• Degradation of photosystem II
• Formation of leghemoglobin in Rhizobium
• A protein to bind O2 and bring it to respiration chain
• Increased respiratory activity around cell membrane
• This facilitates utilization/reduction of O2 to H2O.
國立交通大學生物科技學系 蘭宜錚老師
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Mo‐dependent Nitrogenase
• Nitrogenases are multi‐subunit polypeptide complex coded by the nif genes
• The most studied type of nitrogenase is Mo‐dependent nitrogenase
• Other types of nitrogenases include Vanadium and iron only
• Different nitrogenases produce different levels of byproduct H2.
• Mo‐dependent Nitrogenase consists of 2 proteins
• Dinitrogenase (also called MoFe protein or component I)
• Catalyzes reduction of N2 to 2 NH3 and H2
• Dinitrogenase reductase (also called Fe protein or component II)
• Transfers reducing power (e‐) from reduced ferredoxin to dinitrogenase
Mo‐dependent Nitrogenase
Dinitrogenase reductase (Fe protein)
•
•
•
2 ATP consumed for every 1 e‐ transferred
Reduced ferredoxins (differs between organism) are used as reducing power to transfer e‐
Hydrolysis of ATP causes a conformational
change of the Dinitrogenase reductase and
drives its association with dinitrogenase
Fdred
“Dinitrogenase
reductase”
Dinitrogenase (MoFe protein)
•
•
Catalytic cycle is completed by sequential
transfer of e‐ from Dinitrogenase
reductase. En, where n is the number of e‐
donated by Dinitrogenase reductase.
Contains 2 different novel FeS complex
• P cluster
• FeMo‐co – contains Mo atom and a
homocitrate.
國立交通大學生物科技學系 蘭宜錚老師
“Dinitrogenase”
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Mo‐dependent Nitrogenase
A closer look at Mo‐dependent nitrogenase from Azotobacter
vinelandii.
FeMo‐co:
Interestingly, homocitrate comes from plants symbiotic with the Rhizobium microbe. Nitrogen availability and impact to society
• NH3 ammonia is the most important nitrogen compound that almost all life could use
and is vital for crop production. However, biological N2 fixation is limited in rate as
N=N is extremely stable.
• In 1909 – Fritz Haber invented the direct chemical synthesis of NH3 from N2 + H2 in
lab. immediately German chemical company BASF bought the process and tried to
scale it up. BASF engineer Carl Bosch was successful to scale the Haber process to
industrial scale.
• In 1913 – BASF manufactured NH3 on commercial scale with astonishing rate. This
process remained in use today.
• World population increased from 1.6 billion to 7 billion from 1900 to current day
Fritz Haber
Nobel prize 1918
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Carl Bosch
Nobel prize 1931
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Billion dollar bioengineering challenge!
• Haber‐Bosch process required intensive amounts of energy (High pressure and
temperature). Furthermore, Currently H2 is derived from CH4 (Natural gas) steam
reforming.
• Biological Nitrogenase reaction can occur at ambient temperature and pressure,
which would be more eco‐friendly.
• The KEY challenge is to improve nitrogenase reaction rate and condition.
• What do you think an ideal biological system would be to replace Haber‐Bosch
process?
Nitrate (NO3‐)reduction to nitrite (NO2‐)
•
The ability to reduce nitrate to ammonia is common to virtually all plants, fungi, and bacteria.
•
The first step, reduction of nitrate (NO3‐) to nitrite (NO2‐) is catalyzed by nitrate reductase.
•
The eukaryotic enzyme contains bound FAD, molybdenum, and a cytochrome b5.
•
The enzyme carries out the overall reaction:
•
Where electrons are transferred from:
NAD(P)H  FAD  cyt b5  molybdenum  NO3‐
•
The molybdenum cofactor is typically arranged in molybdopterin
國立交通大學生物科技學系 蘭宜錚老師
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Nitrite (NO2‐) reduction to Ammonia (NH3)
•
Reduction of nitrite to ammonia is carried out in three steps by
one enzyme, nitrite reductase.
•
Higher plants, algae, and cyanobacteria use ferredoxin as the
electron donor in this six‐electron reaction.
•
This enzyme contains one Fe4S4 center and one molecule of
siroheme, a partially reduced iron porphyrin.
How ammonia is incorporated into metabolism
Reactions in assimilation of ammonia and major fates of the fixed nitrogen:
1.Glutamate dehydrogenase
2.Glutamine synthetase
3.Asparagine synthetase
4.Carbamoyl phosphate synthetase
5.Glutamate synthase.
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