20 - Nitrogen Fixation

Question 1

Describe the nitrogen economy, starting at atmospheric nitrogen gas or nitrate in the soil.

Answer 1

• Nitrogen gas is fixed by diazotrophs in soil into ammonia
• Ammonia is assimilated into biomolecules
• Biomolecules are decomposed back into ammonia, mostly through decomposition
• Ammonia is nitrified into nitrite, nitrite is converted to nitrate.
• Nitrate can be dentrificated into atmospheric nitrogen gas
• Soil nitrate is converted into nitrite by nitrate reductase
• Nitrite is ammonified into ammonia via nitrite reductase. ;
• Ammonia can be used as above

Question 2

The Haber process converts gaseous nitrogen into ammonia using pressure and heat. How does this occur biologically?

Answer 2

WIth tons of ATP. There is some futile cycling with H generation.

H2 can be recycled to generate e- and H+ for the reaction. ATP is derived primarily from carbohydrate metabolism.

This is a very energy expensive reaction (high activation energy), though it is exergonic (gives off heat).

Question 3

What are some special characteristics of nitrogen fixing organisms?

Answer 3

Bacteria do the fixing, often in components of larger plants (eg. below).

Alfalfa have rhizobium, nodules on their roots which create anaerobic conditions so that leghemoglobin can sequester oxygen gas (take it away).

Question 4

Describe the nitrogenase complex

Answer 4

Essential for nitrogen fixation

2 proteins

• Fe-protein (component II, nitrogenase reductase)
• MoFe-protein (component I, nitrogenase)

Component II contains iron-sulfur clusters and 2 ATP binding sites.

Component I has redox centers (P-cluster and FeMo cofactor). Generates reducing equivalents. NItrogen fixation occurs on component I.

Question 5

What is a P-cluster?

Answer 5

Found in component I of nitrogenase complex

[4Fe-4S] and [4Fe-3S] liganded via cystine residues in protein.

Question 6

What is a FeMo cofactor?

Answer 6

Found in component I of nitrogenase complex.

Bridged [4Fe=4S] and [Mo-3Fe-3S] clusters

Question 7

What are some special characteristics of nitrogen fixing organisms?

Answer 7

Bacteria do the fixing, often in components of larger plants (eg. below).

Alfalfa have rhizobium, nodules on their roots which create anaerobic conditions so that leghemoglobin can sequester oxygen gas (take it away).

NItrogen fixation is rapidly inactivated in the presence of oxygen. Organisms have mechanisms to exclude oxygen:

• Anaerobic conditions (under soil surface)
• Leghemoglobin binds oxygen gas
• Formation of specialized cell wall to exclude oxygen gas

Question 8

What is a FeMo cofactor?

Answer 8

Found in component I of nitrogenase complex.

Bridged [4Fe=4S] and [Mo-3Fe-3S] clusters

Question 9

What are the three steps of nitrogen reduction?

Answer 9

Involves the segmented passing of electrons:

Fe-S cluster to P cluster to Fe-Mo

1. 2 electrons acquired onto nitrite to make diimine
2. 2 more electrons acquired onto diimine to make hydrazine
3. Hydrazine nitrogen bond broken to generate 2 ammonia

Question 10

Describe energy consumption during nitrogen fixation

Answer 10

• ATP hydrolysis causes a conformational change in Fe-protein permitting the electron transfer to the MoFe protein. This has to occur eight times for each ammonia (2 ATP per time, 1 for each subunit). ATP hydrolysis is involved in conformational change to move Fe-S cluster closer to MoFe

Question 11

Why would we want to study nitrogen fixation?

Answer 11

To genetically modify plants to reduce requirement for fertilizer.

Question 12

Why would we want to study nitrogen fixation?

Answer 12

To genetically modify plants to reduce requirement for fertilizer.

Question 13

What four reactions assimilate ammonia into organic nitrogen species?

Answer 13

Most is assimilated via glutamate (involved in elimination of NH3) or glutamine

• Carbamoyl phosphate and asparagine are two other molecules that ammonia can be incorporated into

Question 14

Describe the enzyme that participates in the reductive aminatino of alpha ketoglutarate to form glutamic acid and the significance of this reaction

Answer 14

Glutamate dehydrogenase.

• Assimilation of ammonia into glutamate
• Requires very high concentration of ammonia, used by plants in presence of NH3.
• Reversible reaction occurs more in animals
• Requires NADH (catabolic) or NADPH (biosynthetic)
• NADH is more commonly used for mitochondrial enzyme (primarily in glutamine degradation)

Question 15

Describe the enzyme that participates in the transamination of glutamate to glutamine

Answer 15

Glutamine synthetase

• Irreversible
• Requires ATP (synthetase, not synthase)
• Optimal at LOW NH4 levels (opposite of glutamate dehydrogenase)
• Amide nitrogen is important in synthesis of other amino acids (where it becomes the alpha amino N), purine and pyrimidine nucleotides and amino sugars (eg. carboxylic acids in TCA cycle).
• REGULATORY SITE OF AMINO ACID SYNTHESIS
• Most important enzyme for ammonia assimilation in most species

Question 16

Glutamine synthetase in E coli responds to a ratio of alpha ketoglutarate to glutamine. Explain this and then give the steps when alpha ketoglutarate is dominant.

Answer 16

When alpha ketoglutarate is high, the pathway is stimulated to convert AK to glutamine.

• Alpha ketoglutarate to glutamate via aminotransferase glutamate dehydrogenase reaction
• Glutamate to acylphosphate intermediate and then glutamine with glutamine synthetase

Question 17

How is glutamine synthetase regulated?

Answer 17

Activity of enzyme is altered by covalent modification (adenylylation)

Question 18

How is glutamine synthetase regulated COVALENTLY?

Answer 18

Activity of enzyme is altered by covalent modification (adenylylation). This is the most important regulatory site of amino acid synthesis.

• Metabolic state of cell regulates conversion of active/inactive forms
• Increased levels of ATP or aKG promote the uridylation of PII and deadenylation of GS to increase its activity (protein synthesis is promoted with more AA being synthesized).
• Adenylation on Tyr residue near the active site gives less active enzyme
• Increases in Gln and Pi promote the de-uridylation of PII and adenylation of GS to decrease its activity
• Regulatory adenylyltransferase with PII cofactor is reversibly uridylylated

Question 19

How is glutamine synthetase regulated allosterically?

Answer 19

Cumulative feedback inhibition (enzyme has 9 different allosteric effectors that are triggered by most amino acids and amino acid metabolites (that contain nitrogen)).

This is a graded response of inhibition (from binding of effectors), and binding on different sites on each monomer of the synthetase hexamer.

Each gives incremental inhibitino of activity but all 9 gives total inhibition of activity.