Nitrogen Cycle

Question 1

What’s the problem with the nature of atmospheric nitrogen?

Answer 1

N₂ (atmospheric nitrogen) has a triple bond that is extremely INERT!

== very unreactive; we CANNOT use atmospheric nitrogen directly in biosynthesis!

Question 2

What is the nitrogen cycle?

Answer 2

The conversion of nitrogen through various oxidation states in various organisms

Question 2

What is the “solution” to the problem of atmospheric nitrogen being unreactive?

Answer 2

Nitrogen Fixation!

== reducing atmospheric nitrogen (N₂) to a form usable by life (ammonia = NH3)

Question 3

What are the 4 main processes in the nitrogen cycle (list them)?

Answer 3

1) Nitrogen Fixation
2) Nitrification
3) Denitrification
4) Assimilation

Question 4

What is nitrogen fixation?

Answer 4

The reduction of atmospheric nitrogen (N₂) to biologically available ammonia (NH₃)

== Carried out by nitrogen fixing bacteria (Diazotropes)

Question 5

What is nitrification?

Answer 5

The oxidation of ammonia to nitrite(NO₂^-) and nitrate (NO₃^-)

== Carried out by nitrifying bacteria

Question 6

What is denitrification?

Answer 6

The reduction of nitrite(NO₂^-) and nitrate (NO₃^-) to atmospheric nitrogen (N₂)

== Carried out by denitrifying bacteria

Question 7

What kind of conditions are required for denitrification?

Answer 7

ANAEROBIC conditions!

== to allow for NO₃^- to act as an electron acceptor rather than oxygen!

Question 8

What is assimilation?

Answer 8

The reduction of nitrite(NO₂^-) and nitrate (NO₃^-) to ammonia (NH₃)

== catalyzed by two enzymes: (1) Nitrate reductase, (2) Nitrite reductase

== carried out in PLANTS + MICROORGS!

Question 9

Overall, how does assimilation occur?

Answer 9

Assimilation occurs via 2 reduction rxns

Red. 1) Nitrate ——> Nitrite (via nitrate reductase)

Red. 2) Nitrite ———> Ammonia (via nitrite reductase

Question 10

In assimilation, how many electrons need to be transferred?

Answer 10

EIGHT electrons need to be transferred to nitrate (NO₃^-)

Question 11

What are the cofactors used in nitrAte reductase?

(give in order of electron transfer)

Answer 11

Source of electrons = NADH

1) SH (sulfur atom)
2) FAD
3) CytB-557
4) Mo-Co (molybdenum cofactor)

Question 12

Explain the process of electron transfer for the first reduction reaction in assimilation:

Answer 12

Enzyme = Nitrate reductase

Electron Source = NADH

1) NADH donates electron to sulfur atom of the enzyme

2) Reduced sulfur atom donates electron to FAD

3) Reduced FAD donates electrons to CytB-557

4) Reduced CytB557 donates electrons to Mo-Co (molybdenum cofactor)

5) Reduced Mo-Co donates TWO electrons to nitrate (NO₃^-)! == nitrite(NO₂^-)

Question 13

Explain the process of electron transfer for the second reduction reaction in assimilation:

Answer 13

Enzyme = Nitrite Reductase

Electron Source = Ferredoxin (if photosynthetic organism)

1) Reduced ferredoxin donates electrons to 4Fe-4S clusters

2) Reduced 4Fe-4S clusters donates electrons to Siroheme

3) Reduced Siroheme donates SIX electrons to nitrite (NO₂^-) == generate ammonia (NH₃)

Question 14

What are the cofactors used in nitrIte reductase?

(give in order of electron transfer)

Answer 14

1) 4Fe-4S cluster

2) Siroheme

Question 15

What is siroheme?

Answer 15

A cofactor used in electron transport that is almost identical to heme except it has a bunch of carboxylic acid substituents attached to the porphyrin ring!

Question 16

For the second reduction reaction in assimilation catalyzed by nitrtie reducatase, what is the source of electrons?

Answer 16

Depends on type of organism:

1) Photosynthetic plants/microorgs = FERREDOXIN(red)

2) NON-photosynthetic microorgs = NADPH!

Question 17

How is ferredoxin initially reduced to then be able to enter assimilation?

Answer 17

Ferredoxin is initially reduced via the light dependent rxns in the chloroplasts!

Question 18

What are the 2 ways nitrogen fixation can occur?

Answer 18

1) Industrially (Haber Process)

2) Naturally (Bacteria: Diazotrophs)

Question 19

What is the Haber process?

What is it used for, what does it require?

Answer 19

The Haber process is a method of carrying out nitrogen fixation on an industrial scale

It involves pumping hydrogen gas (H₂) into N₂ at very high temperature and pressure to create ammonia (NH₃)

Overall Eqn: N₂ + 3H₂ —–> 2NH₃

Purpose: Create ammonia to supply into fertilizer to support crops around the world (haber process supports enough crops for 1/3 of the world)

Expense: Uses A LOT of energy: 1-2% of the world’s annual energy supply goes to the haber process!

Question 20

Overall, how is natural nitrogen fixation carried out? What does it require?

(not the detailed process)

Answer 20

Natrual nitrogen fixation is carried out by nitrogen fixing bacteria DIAZOTROPHS

—> Utilize an enzyme complex called NITROGENASE!

—> Requires input of 16 ATP!

Question 21

Why does nitrogen fixation require so much energy?

Answer 21

Due to its high activation energy!

–> Atmospheric nitrogen has a triple bond that requires A LOT of energy in order to break! == needs a lot of energy to get over the initial hump!

Question 22

Is nitrogen fixation endergonic or exergonic?

Answer 22

EXERGONIC

(BUT it still happens quite slowly due to its large activation energy)

Question 23

Explain the speed of nitrogen fixation and the factors contributing to these kinetics:

Answer 23

Nitrogen fixation is SLOW!

== Due to high activation energy caused by the very stable triple bond in atmospheric nitrogen!

Question 24

What is the overall rxn expression for nitrogen fixation?

Answer 24

Question 25

How much ATP is consumed in nitrogen fixation?

Answer 25

16 ATP per N₂ molecule!

Question 26

What is the nitrogenase complex?

Answer 26

A massive complex of proteins that are capable of reducing N₂ to NH₃ in the presence of ATP! == Enzyme complex used in nitrogen fixation!

Question 27

What are the main components of nitrogenase?

Answer 27

Two main components: 1) **Dinitrogenase Reductase** (Made of 2 identical subunits) 2) **Dinitrogenase** (Made of a tetramer of 2α and 2β subunits)

Question 28

How do the main components of nitrogenase differ?

Answer 28

Differ in their structure/electron carriers! **Dinitrogenase reductase** == Has one 4Fe-4S cluster + ATP binding sites **Dinitrogenase** = Has one P-cluster and one FeMo Cluster

Question 29

Nitrogenase function requires _____________ conditions because...

Answer 29

Nitrogenase function requires **ANAEROBIC** conditions because... == The enzyme carrier cofactors in the enzyme complex are extremely sensitive to oxygen; they burst in the presence of oxygen!

Question 30

What is the order of electron flow through the electron carriers in nitrogenase?

Answer 30

1) 4Fe-4S cluster (dinitrogenase reductase) 2) P-cluster (dinitrogenase) 3) FeMo Cluster (dinitrogenase)

Question 31

What do the components of the nitrogenase complex catalyze?

Answer 31

**Dinitrogenase Reductase** (1) Catalyzes transfer of 8 electrons to dinitrogenase (2) Catalyzes the binding + hydrolysis of ATP along with the release of H+ **Dinitrogenase** (1) Catalyzes the transfer of 6 electrons to N₂ forming NH₃ (2) Catalyzes the transfer of 2 electrons to H+ forming H₂

Question 32

Explain the entire process of nitrogen fixation via nitrogenase:

Answer 32

1) Electrons from 4CoA + 4 Pyruvate transfer to 8 Ferredoxin molecules == 8 Fd_red 2) 8 Fd_red transfer their 8 electrons to the 4Fe-4S cluster in 8 dinitrogenase reductases, reducing the complexes 3) Reduction of dinitrogenase reductase triggers binding of 16 ATP to the 8 complexes 4) Hydrolysis of ATP allows for transfer of 8 electrons to 8 dinitrogenase complexes from the 4Fe-4s cluster in the 8 dinitrogenase reductase complexes 5) The 8 electrons transferred to 8 dinitrogenases travel through the P cluster and FeMo cluster in the complexes 6) The 8 reduced dinitrogenases transfer 6 electrons from their FeMo Clusters to N₂ forming NH₃ and 2 electrons from their FeMo are transferred to H+ to form H₂

Question 33

What are the initial sources and acceptors of electrons in the nitrogenase complex process

Answer 33

**Initial source of electrons** = CoA + Pyruvate **Initial Acceptor** = Ferredoxin OR Flavodoxin!

Question 34

The mechanism of which enzyme in nitrogen fixation is not yet well understood?

Answer 34

The mechanism of **dinitrogenase** is not yet well understood!

Question 35

Explain the sybmiotic relationship diazotrophs are involved in:

Answer 35

Diazotrophs (nitrogen fixing bacteria) are involved in a symbiotic relationship with leguminous plants! --> The diazotrophs colonize the root nodules of these plants **Diazotrophs provide**: --> Critical *AMMONIA* to the plant (has they carry out fixation) **The plants provide**: (1) *Leghemoglobin* for protection against oxygen interference with electron carriers in nitrogenase (bacteria get coated in leghemoglobin) (2) *A source of carbs* + energy sources for the bacteria

Question 36

What is leghemoglobin?

Answer 36

Leghemoglobin == an oxygen-binding heme protein --> Plants produce leghemoglobin and so diazotrophs colonizing their root nodules get coated in it! == Leghemoglobin binds all available oxygen so that it cannot interfere with nitrogen fixation by disrupting the critical electron carriers in nitrogenase

Question 37

What are some similarities between assimilation and fixation? (4)

Answer 37

1) Both are aimed at producing ammonia 2) Both are electron transfer processes 3) Both use some form of Molybdenum cofactor at some point 4) Both involve the use of multiple redox cofactors

Question 38

What are some differences between assimilation and fixation? (3)

Answer 38

Assimilation: 1) Uses nitrates and nitrates to make ammonia 2) Does NOT require ATP 3) Electrons come from NADPH or Ferredoxin Fixation: 1) Uses atmospheric nitrogen to make ammonia 2) Requires A LOT of ATP! 3) Electrons come from pyruvate + CoA

Question 39

Once ammonia is made via assimilation or fixation, what must happen?

Answer 39

The nitrogen in ammonia must be incorporated into biomolecules to allow for biosynthesis!

Question 40

What are the 2 entry point for nitrogen in ammonia into biomolecules?

Answer 40

Glutamate + Glutamine!

Question 41

Explain the first step in the process of incorporating ammonia into biomolecules

Answer 41

Glutamate + Ammonia -------> Glutamine! == Occurs via enzyme **Glutamine Synthetase** which catalyzes two steps: (1) Glutamate is phosphorylated at its side chain COO- grp == yields **γ-glutamyl phosphate** (2) Ammonia then comes in and displaces the phosphate grp (released as Pi), forming **Glutamine** --> Glutamine can now go serve as a substrate for the synthesis of other biomolecules

Question 41

Ammonia created from nitrogen fixation/assimilation will be incorporated into biomolecules by creating ______________ using ________________

Answer 41

Ammonia created from nitrogen fixation/assimilation will be incorporated into biomolecules by **creating GLUTAMINE** through the **use of GLUTAMATE + ammonia**

Question 42

Why is the synthesis of glutamine a major point of control?

Answer 42

Because the glutamine made from ammonia that came from nitrogen fixation is what controls the amount ability of other biosynthesis pathways to take place! == Glutamine is a precursor in MANY biosynthesis pathways so its availability can control whether or not these pathways occur!

Question 43

How is the concentration of glutamine controlled?

Answer 43

Controlled by the activity of glutamine synthetase! (which is allosterically regulated)

Question 44

Explain the structure of glutamine synthetase and where all substrate binding sites are:

Answer 44

**Glutamine synthetase** == 12 subunits; made up of 2 stacked hexameric rings ---> Substrate binding sites (for ATP and glutamate) are found at the interfaces between subunits!

Question 45

Why does the structure of glutamate synthetase lend itself to allosteric regulation?

Answer 45

Because of the interfaces between subunits, this allows for inter-subunit communication in which binding of an allosteric regulator to one subunit can be communicated to all the other subunits!

Question 46

What are the allosteric regulators of glutamine synthetase?

Answer 46

**These are all allosteric INHIBITORS** (1) ALL end products of biosynthesis pathways that START with glutamine! (2) Alanine, Serine, and Glycine **The end products:** 1) CTP 2) AMP 3) Histidine 4) Carbamoyl Phosphate 5) Glucosamine-6P 6) Tryptophan

Question 47

The negative feedback on glutamine synthetase allows for...

Answer 47

The negative feedback on glutamine synthetase allows for... == sensing of a cell's nitrogen levels!

Question 48

After incorporation of ammonia into glutamine, what else do plants + bacteria do that we can't? WHY?

Answer 48

Plants + bacteria use **Glutamate Synthase** to convert the glutamine into glutamate! **Purpose** = To regenerate glutamate; preventing the overuse of glutamate + shift the net rxn towards glutamate production rather than usage

Question 49

Explain the second rxn plants + bacteria carry out after glutamine synthesis:

Answer 49

(1) Glutamate + Ammonia ----> Glutamine **(2) Glutamine + αKG + NADPH -------> 2 Glutamate + NADP+** == This second rxn uses **Glutamate Synthase** (humans do not have)

Question 50

In plants and bacteria, what is the NET RXN EQN for the reactions carried out by glutamine synthetase followed by glutamate synthase in the process of incorporating ammonia into biomolecules?

Answer 50

The net rxn is essentially the combination of αKG with ammonia to produce glutamate! (Humans can't do this because we lack the enzyme; we convert glutamine to glutamate using transamination rxns)