Lecture 1: Nitrogen Cycle and Fixation Flashcards
How does the nitrogen cycle work?
- Nitrogen is inert in our atmosphere (it makes up 78% of the total volume).
- Nitrogen is fixed by bacteria in the soil and root nodules of legumes.
- The nitrogen can be fixed into ammonium.
- The ammonium is converted into nitrite and then nitrate by nitrifying bacteria.
- The nitrates can be broken down by denitrifying bacteria. The nitrate can then decompose and be placed back into the atmosphere.
- Nitrates are assimilated by plants and enter the food chain.
- Nitrogen found in organisms will eventually decompose and go back into the soil.
How does nitrogen fixation work thermodynamically?
Overall, the reaction is favourable.
• It is difficult because dinitrogen is thermodynamically inert.
• When different intermediates are created, the enzyme can make a thermodynamically favourable reaction.
• Nitrogen and hydrogen form diazene (unfavourable).
• This is added to hydrogen to make hydrazine (favourable).
• Hydrazine is added to hydrogen and 2 ammonia forms (favourable).
How does nitrogenase work? How do the cofactors work?
- One of the main issues with dinitrogen is that it is kinetically inert. It makes a very poor metal ligand. It is non-polar and it has tightly bound σ and π electrons.
- The reaction costs a lot of ATP (16 molecules) compared to carbon fixation.
- However, ATP hydrolysis is not required for the overall thermodynamics of the nitrogenase reaction. The reaction is still feasible.
- Nitrogenase requires a lower potential electron donor than NAD(P)H, which has a reduction potential of -0.34V.
- Ferredoxin binds to a [4Fe-4S] cluster. It can carry 1 electron and it has a reduction potential of -0.45V.
- Flavodoxin binds to FMN. It also carries 1 electron and has a reduction potential of -0.45V.
- Nitrogenase is oxygen sensitive, components are irreversibly inactivated by it. This is because it is full of low potential redox centres, it is very sensitive.
- Nitrogenase works very slowly, at about 5 s-1 per electron. The enzyme is therefore present at very high concentrations in nitrogen fixing bacteria.
How can we measure nitrogenase activity?
- Many molecules containing multiple bonds can be reduced by nitrogenase such as acetylene, carbon monoxide and cyanide.
- These molecules are used as probes.
- Acetylene is used to measure activity. Ethene is produced as a product, which is readily quantified by gas chromatography.
What is the structure of nitrogenase? Draw a diagram
Nitrogenase consists of two separate proteins: dinitrogenase reductase (Fe-protein) and dinitrogenase (MoFe-protein).
• Electron donors are ferredoxin or flavodoxin.
• Electron flow is: electron donor -> dinitrogenase reductase -> dinitrogenase -> dinitrogen.
• The nitrogenase P cluster probably acts as an electron carrier.
• There is some evidence that ligand exchange and structural rearrangement occur during physiological oxidation of the cluster.
• The cluster can be though of 2 Fe-4S clusters sharing an S atom.
• The FeMo cofactor also has an important role. We have a very high-resolution structure of it.
What is the Fe protein cycle?
• The Fe-S cluster in the Fe protein is represented by a cube.
• In the MoFe protein, the P cluster is represented by a square and the Mo cofactor by a diamond.
• The Fe protein must be reduced by an external electron donor on the left-hand side on the scheme.
• The Fe protein only hydrolyses ATP when complexed with the MoFe protein.
1) Fe protein reduced (2ATP) forms a complex with MoFe protein.
2) Electron transfer from Fe to MoFe is coupled to hydrolysis of ATP.
3) The phosphate are released. This is the RDS, when the physiological external electron donor flavodoxin is used.
4) Dissociation of the oxidised, ADP-bound, Fe protein.
5) Re-reduction of Fe protein.
6) Exchange of 2ATP for 2ADP bound to the Fe-protein.
7) The cycle is repeated 8 times to affect the reduction of one molecule of dinitrogen.
How do electrons move in the nitrogenase complex?
Electrons must move from the [4Fe-4S] complex to FeMoco.
• The maximum separation which allows electron transfer is about 14 Angstroms.
• The distance between [4Fe-4S] and FeMoco is more than 14 Angstroms.
• [4Fe-4S] is 13 Angstroms from the P-cluster, which is itself 14 Angstroms from FeMoco.
• The structure suggests that the order is [4Fe-4S] P-cluster FeMoco.
• Each Mo-Fe protein heterodimer is able to interact independently with Fe protein.
How does Fe-protein work?
The Fe-protein has 2 conformations: open and closed.
• The open conformation is for when Fe is not bound to the MoFe protein or for when it is ADP-bound and in complex with MoFe protein.
• The closed conformation is for when Fe is in complex with MoFe and binding an ATP. It can be crystalized with a non-hydrolysable ATP analogue or a transition state analogue.
• The closed form allows ATP hydrolysis by stabilising the catalytically competent form of the nucleotide binding site. Two essential catalytic residues: lys10 and asp129 are brought close enough to the nucleotide to participate in catalysis.
How does the Fe-MoFe complex work?
The two proteins complex together in order to form the full nitrogenase enzyme.
• The tightest, most symmetrical fit was found in the ADP-ALF4- loaded complex.
• A loose and asymmetric fit was observed in the ADP loaded complex.
• The MoFe protein appears to act as a rigid body. Binding of Fe causes no significant changes in the MoFe protein.
• The closed conformation of the Fe-protein has much greater shape complementarity to the MoFe-protein than the open conformation.
• The MoFe protein stabilises the closed state of the Fe protein.
• The 4Fe-4S cluster is pushed towards the apex of the Fe-protein in the closed structure. This reduces the electron transfer distance to the P cluster.
• The change to the closed conformer induces ATP hydrolysis. This explains why ATP is not hydrolysed by free Fe-protein.
• The ADP bound state favours the open conformer. This leads to a loosening of the interaction between the Fe-protein and the MoFe protein.
• This explains the dissociation of the ADP bound form of the Fe-protein from the complex.
What is the super reducing electrons model?
- This model proposes that ATP hydrolysis is used to lower the reduction potential of the electrons delivered by the Fe-protein.
- Changes in the conformation of the Fe protein change the environment, and thus reduction potential of the 4Fe-4S cluster by about -200mV on complex formation with the MoFe protein.
- However, very low potential chemical reductants do not support dinitrogen reduction.
What is the clock model?
- Proposes that ATP-driven conformational cycle undergone by the Fe protein is used to limit the rate of electron transfer to the MoFe protein.
- This would mean that electrons are only injected into the nitrogen reduction reaction at appropriate times.
What is the ‘deficit spending’ model?
- Proposes that the ATP-bound form of the Fe protein drives a conformational change in the MoFe protein that drives an electron from the P cluster to the FeMoco.
- This electron is replaced by a transfer from the Fe protein to the P cluster.
- Such a conformational gating of electron flow to the active site could function to make the electron transfer irreversible.
- The model is supported by kinetic analysis of electron flow between the cofactors. The P cluster is already fully reduced in the resting state, so it can’t initially receive electrons from the Fe protein.
- No conformational change is seen in MoFe protein crystal studies, which is a point against this model. However, electron transfer to FeMoco is only seen if the Fe protein has ATP bound, not ATP analogues. So, the relevant state has probably not yet been crystallised.
How does catalysis at FeMoco work?
- We also have a model for the MoFe cofactor binding to nitrogen.
- Only the face of the MoFe cofactor involved in nitrogen binding is shown.
- Reductive elimination of the bridging hydrides as dihydrogen leaves results in a two-electron reduction of the cofactor, which is now super-reduced and more reactive towards dinitrogen.
- The nitrogen for hydrogen exchange is readily reversible.
