Enzymology Of Oxygen And Nitrogen

Question 1

How can nitrogen be fixed?
What are the only organisms that can fix nitrogen?
Nitrogen fixation is a strictly what process?

Answer 1

Through the Haber Bosch process,
Through lightning,
Through biological means (Nitrogenase).
Bacteria are the only organisms that can fix nitrogen.
Anaerobic process due to the oxygen sensitive nature of Nitrogenase.

Question 2

What reaction does Nitrogenase catalyse?

Answer 2

N2 + 8H+ + 8e- +16MgATP –> 2NH3 + H2 + 16MgADP + 16Pi

Question 3

Nitrogenase is made up of two proteins, what are these proteins?

Answer 3

One is a heterotetramer (the MoFe protein) and is made up of two α subunits and two β subunits.
The other is a dimer (γ2) (the Fe Protein).

Question 4

Describe briefly the function of the (γ2) dimer in Nitrogenase.

Answer 4

This Fe Protein is a dimer of identical subunits which contains one Fe4S4 cluster and weighs approximately 60-64kDa. The function of the Fe Protein is to transfer electrons from the reducing agent, such as ferrodoxin or flacodoxin, to the MoFe protein. The transfer of electrons occurs via the binding and hydrolysis of ATP. This hydrolysis also causes a conformational change which brings the Fe protein and MoFe protein closer together.

Question 5

Briefly describe the components of the heterotetramer (α2β2).

Answer 5

The MoFe protein weighs approximately 240-250kDa. This protein contains two iron-sulphur clusters known as P-clusters located at the interface between the α and β subunits. It also contains FeMo cofactors within the α subunits.

Question 6

What form of Nitrogenase is preferentially expressed but what can alternatively be expressed?
Briefly describe what this alternative version of Nitrogenase can do?

Answer 6

Molybdenum Nitrogenase is preferentially expressed but vanadium Nitrogenase can also be expressed.
Vanadium nitrogenases can also fix nitrogen gas into ammonia however they can also fix carbon monoxide into alkanes via a process similar to Fischer-Tropsch synthesis.

Question 7

What are Iron-Sulphur clusters?

Answer 7

These are ancient cofactors composed of Iron and Sulphur. These clusters are extremely sensitive to oxygen (often being protected by proteins). The clusters are common in electron transfer pathways where single electron transfers are common. Fe-S clusters are found in Quinone dehydrogenases such as NADH dehydrogenase.

Question 8

Where is the Iron-Sulphur (Fe4S4) cluster located?

How many MgATP binding sites does the Fe Protein possess?

Answer 8

It is located at the dimer interface in the Fe Protein. Two cysteines are in each monomer that bind Fe. The cluster can be reduced by flavodoxin, ferrodoxin.
Possesses two MgATP binding sites.

Question 9

What gene is involved in the Fe Protein?

Answer 9

The NifH gene.

Question 10

What do the P-clusters do in the MoFe protein? Describe their structure and confirmation along with their oxidation states.

Answer 10

These transfer electrons to the FeMo cofactor. The core (Fe8S7) of the P-cluster takes the form of two (Fe4S3) cubes bonded by a central sulphur atom. Each P-cluster is covalently linked to the MoFe protein by six cysteine residues. P-clusters have several oxidation states: 0 (PN), +1 (P semi-Ox), +2 (P OX), +3 (P OX2), +4 (P Superox)

Question 11

Describe the FeMo protein cofactor.

Answer 11

The cofactor has a formula of Fe7S9Mo-C with each protein being comprised of two non-identical clusters: Fe4S3 and MoFe3S3, these being linked by 3 sulphide ions. They are linked to the α subunit by a cysteine and histidine residue.

Question 12

Crystal structure imaging of the FeMo cofactor at what resolution has shown what?

Answer 12

At a resolution of 1.1Å has shown the active site of the cofactor.

Question 13

What is the overall cycle of the MoFe Protein-Fe Protein interaction/dissociation?

Answer 13

Fe protein binds MgATP
Forms a complex with the MoFe protein,
Transforms electrons, ATP hydrolysis,
Fe protein dissociates,
Nucleotides are exchanged.

Question 14

The Fe protein can be reversibly reduced by what?

The MoFe protein has how many Fe association sites?

Answer 14

By 1e-

2 Fe protein association sites.

Question 15

How many MgATP are hydrolysed for every Fe protein-MoFe protein association/dissociation?

Answer 15

2 MgATP are hydrolysed.

Question 16

What model describes the reduction of dinitrogen?

Answer 16

The Lowe Thorneley model.

Question 17

Describe, briefly, the Lowe Thorneley model.

Answer 17

The E0 state of Nitrogenase undergoes three reductions whereby three hydrogens are bound to Mo. Two hydrogens are then removed and N2 binds to form E3N2H. This is reduced again to form E4-N-NH2. At a pH of 0 or 14 N2H4 dissociates via acid/alkali quenching. Otherwise E4N2H4 is reduced again to form E5-N-NH3+, with NH3 dissociating with another reduction.

Question 18

At what state does acetylene (C2H2) and dinitrogen (N2) bind to the MoFe protein?
What occurs if there is not enough ATP?

Answer 18

Acetylene binds at the E1 state, dinitrogen binds at the E3 state. If there is not enough ATP the E3 state will not be reached and N2 will not be fixed.

Question 19

Where do protons required for reduction originate?

What residues bind the FeMo protein to the Nitrogenase enzyme?

Answer 19

From water surrounding FeMo-cofactor and homocitrate.
His442 and Cys275
Hydrogen bonding occurs from His195, which also acts as a proton donor.

Question 20

What is an essential element for life with a triple bond that is very hard break?
What is the dissociation energy of this bond?
When the fixed form is expended, what form is turned to?

Answer 20

Nitrogen is an essential element with a hard triple bond. It has a dissociation energy of 945kJ/mol.
When nitrate is expended, the gaseous form is turned to.

Question 21

Give three reactive oxygen species examples.

Answer 21

Superoxide
Peroxide
Hydroxyl radicals

Question 22

What can free radicals cause?

Answer 22

Cancer,
Ageing,
Possibly other diseases.

Question 23

What enzyme can catalyse superoxide generation.

Answer 23

NADPH.

Question 24

Describe the function of flavonenzymes.

Answer 24

They oxidise a variety of metabolites. They are involved in electron transfers, having important roles in complex I and complex II in oxidative phosphorylation. They are involved in the activation of oxygen.

Question 25

How are hydroxyl radicals removed?

Answer 25

The OH• radical is the most reactive radical species with a half life of 10-9s. Chemical antioxidants in the cell remove OH• ions, such as glutathione.

Question 26

What enzyme catalyses the detoxification of superoxides?
What is the equation for this reaction?
What types of this enzyme are found in what organisms?

Answer 26

Superoxide demutase (SOD) catalyses this reaction.
2O2 –> H2O + O2
Cu and Zn SOD are found in eukaryotic cells,
Mn SOD are found in mitochondria,
Fe SOD and Ni SOD are found in bacteria.

Question 27

How fast is the reaction catalysed by Superoxide demutase?

What directs the superoxide to the active site for this enzyme?

Answer 27

The reaction is very fast (10^8 ms-1).

Positive charges on the enzyme surface direct the ion to the active site.

Question 28

How is hydrogen peroxide detoxified?

Answer 28

It is detoxified by haem containing catalases and peroxidases. Catalases work on a 2 step oxidation/reduction pathway, with typical products being H2O and O2. Peroxidases use an electron acceptor to reduce H2O2 to water. To avoid the Fenton reaction the Haem contains Iron in the Fe^3+ state.