Biochem lec 22: Nitrogen fixation

Nitrogen cycle

N2 to NH4+/amino acids to NO2-/NO3- to N2. (red arrows in anaerobic environments)

Nitrogen fixation

bacteria reduce N2 to NH3 (black arrows)

nitrification

conversion of ammonia to NO3- (purple arrows)

denitrification

conversion of NO3- to N2 (red arrows)

assimilation

plants and microorganisms reduce NO2- and NO3- to NH3 back into the soil (green arrows)

anammox (anaerobic ammonium oxidation)

specialized process where bacterial convert NH3 and NO2- directly into N2 under anaerobic conditions (blue arrows)

Oxidation of nitrogen compounds from oxidized to reduced

NO3-, NO2-, N2, NH3

Nitrogenase complex

N2 to NH3.

has 2 binding sites of ATP/ADP cofactors

Can be oxidized and reduced by 1 electron

central components: dinitrogenase reductase (iron-sulfur cluster), dinitrogenase (dimer)

in anaerobic baterica

dinitrogenase

heterodimer

1. P-cluster (Fe-S center) to transfer e-

2. FeMO cofactor to transfer e-

8 e- transferred from dinitrogenase reductase to dinitrogenase

each turn of the cycle requires 2 ATP

nitrogen fixation procedure

1. 2 ADP bound to dinitrogenase reductase, electron is reduced and held by enzyme

2. 2 ATP replace 2 ADP to induce conformational change

3. Dinitrogenase binds to nitrogenase reductase complex

4. electron transfer

5. ATP hydrolysis and Pi release

6. complex dissociation, repeat

redox reactions in nitrogenase stoichiometry

N2 + 8H+ + 8e- + 16 ATP = 2 NH3 + H2 + 16 ADP + 16 Pi

dinitrogenase reductase catalyzes

transfer of 8e- to dinitrogenase and hydrolysis of ATP with release of protons

dinitrogenase catalyzes

transfer of 6 e- to nitrogen to form NH3 and transfer of 2 e- to protons to form H2

why is nitrogenase only in bacteria

the enzyme is sensitize to oxygenation and requires anaerobic conditions

how do bacteria maintain anaerobic environment?

• burn off O2 with partially uncouples electron transfer

• specialized thick walled called to exclude O2

• plants evolved to provide symbiotic niche for bacteria to fix N2 for them via rhizomes

  • gut bacteria’s ability to fix N2 to NH3

production of amino acids from ammonia

• NH3 made into glutamate and glutamine

glutamine synthesis

enzyme: glutamine synthase converts glutamate to glutamine

1. glutamate + ATP (glutamate kinase) —> intermediate

2. intermediate + NH4+ —> glutamine + Pi + H+

rxn: glutamate + NH4+ + ATP —> glutamine + ADP + Pi + H+

glutamate synthesis

enzyme: glutamate synthase converts glutamine + alpha-KG to 2 glutamates

rxn: alpha-KG + glutamine + NAD(P)H + H+ —> 2 glutamate + NAD(P)+

net reaction of ammonia incorporation into biomolecules

alpha-KG + NH4+ + NAD(P)H + ATP —> glutamate + NAD(P)+ + ADP + Pi

glutamine synthase

in all organisms, primary regulatory point in N2 metabolism. Many things required to shut it off

glutamate synthase

only in bacteria and plants, not animals

where does glutamate come from?

transamination of alpha-ketoglutarate and amino acid catabolism

how is glutamine synthase covalently inhibited?

tryosine in active site needs to be phosphorylated to be active. If this tyrosine is adenylated (via adenylyltransferase and ATP), it won’t work.

P2 regulates adenylytransferase

uridylated P2 (via Uridyltransferase, which is inhibited by glutamine) activates deadenylation

P2 activates adenylation

what amino acids are made from glutamate?

proline, arginine, glutamine

how is proline made from glutamate?

1. glutamate kinase phosphorylates glutamate

2. NADPH + H+ cofactor used to reduce phosphorylated glutamate to a semialdehyde

3. nonenzymatic spontaneous cyclization to P5C

4. pyrroline carboxylate reductase reduces P5C using NADPH + H+ to get proline

how is arginine made from glutamate?

1. gamma-glutamyl phosphate reduced by NADPH or NADH to glutamate gamma-semialdehyde

2. another glutamate is used for transamination

3. acetyl group is removed to make ornithine

4. Urea cycle to turn ornithine into Arginine

proline synthesis from arginine

reverse urea cycle reaction where ornithine is converted to proline