Nitrogen cycle

Describe where nitrogen can be found

• Atmosphere

• Lithosphere - soil and rock

• Ocean

• Constant cycling of these different forms

What are the main processes of the nitrogen cycle?

• N2 fixation

• Denifirification

• Assimilatory and dissimilatory NO3- reduction

• Nitrification

• Some are e- donor requiring, others are e- acceptor requiring

• Ammonia is the most reduced form, nitrate is the most oxidised form

• Different process interconvert the different forms of nitrogen

Describe the redox processes of the denitrification from nitrate to dinitrogen, what organisms can do what?

• Nitrate NO3- is reduced to nitrite NO2- (this is nitrate reduction, for example in E.coli)

• Nitrite is reduced to nitric acid NO

• Nitric acid is reduced to nitrous oxide N2O

• Nitrous oxide is reduced to dinitrogen N2

• Ecoli can only do the first reduction, pseudomonas can do all

What can the cycling between redox states be used as?

• A source of energy as the electrons move from different redox potentials

Describe aerobic respiration vs anerobic respiration (using nitrogen)

Aerobic: Oxygen is the electron acceptor, NAD+ is your first electron acceptor, so E.coli can grown under aerobic conditions

Anaerobic: Nitrogen accepting electrons can be used coupled to electron transport and proton pumping. Nitrate is used as the final electron acceptor, the pathway is very similar, but instead of the final complex transferring e- to oxygen, they are transferred to nitrate to form nitrite, this means E.coli can grow in anaerobic conditions (providing there is nitrate available)

Describe how the interconversion of different nitrogen compounds allows energy release and how this compares to using oxygen as an electron acceptor

• You get energy out by moving to a more positive redox potential, this is displayed in the ETC of aerobic respiration and that of photosynthesis

• The amount of energy we can get out is the difference between the electron donor and the electron acceptor

• NADH has very negative redox potential, oxygen has a very positive redox potential, there is a large difference, this is good as we can get a lot of energy out of this electron transfer

• Nitrate/nitrite does not achieve as high a drop of redox potential, so not as much energy can be harnessed, but we can still get energy for pumping protons

• For oxygen - pumping 10H+, for nitrate - pumping 8H+, this is because we do not have as much energy for proton pumping

Describe nitrate reduction in E.coli

• Need to get nitrate into the cytoplasm as this is where the reduction occurs

• NarK1, uses a pmf to move nitrate into the cell, this so needed when you want to start using nitrate and not already using it as an e- acceptor

• NarK2, antiporter, nitrate comes in and nitrite comes out - somewhat a steady state

• Nitrate reductase is similar to the e- transport proteins - many metal centres and cytochrome bs, so can have electron transport

• Nitrite is toxic so needs to be dealt with

Describe denitrification in pseudomonas

• Takes place in the periplasm - do not have the same transport needs as in E.Coli

• More complicated ETC

• Similar chain in Paracoccus

• Nitrate reductase is present, so can form nitrite from nitrate

• Can also use nitrite itself as an e- acceptor, this forms NO (nitric oxide)

• Can then use NO as an e- acceptor forming N2O, can then use N2O as an e- acceptor and form N2 (nitrogen gas)

• The cyle is not linear. The electron transport chain can be branched at the acceptor end, the electrons from NADH can go onto any of the intermediates that is going to accept electrons, this means different amounts of energy are released, relating to the redox potentials of the compounds

--> can use different compounds depending on what is available in the environment, and can use the same compound as an electron acceptor many times

Why do we reduce nitrite when oxygen is present?

• Oxygen is a great e- acceptor and you get a larger amount of energy out, but organisms still reduce nitrite when oxygen is present

• Whilst it means you get less energy out of the NADH, there is only a limited amount of oxygen, if you can use multiple electron acceptors simultaneously, the amount of energy you can get out per second is higher

• This is important when organisms want to grow quickly in a rich environment that is not limited by the concentration of oxygen

What is the alternative e- donor in aerobic nitrification?

• We could also convert nitrite to nitrate, releasing an electron

• This would allow movement to a higher redox potential (oxygen)

• The change in energy is much smaller than that from NADH, but it is still possible

Describe nitrification

• Occurs in nitrobacteria

• Oxidise nitrite to form nitrate, electrons go via cytochrome C to cytochrome aa3, and then the electrons go onto oxygen to form water

• Can use these electrons to generate a proton motive force and use this to generate ATP as an energy forms

• Using an inorganic compound

• Relatively small amount of energy produced, this limits ATP production (and therefore energy) and growth

• Growth yields are low, so slow growing but can live

Where does nitrification occur?

• In aerobic environments (because oxygen is our electron acceptor) such as water, toxic sediments and estuaries

• In areas rich in organic material, important in sewage treatment, contain lots of nitrogen

What enzyme fixes nitrogen?

Nitrogenase

Describe the structure of nitrogenase

• Nitrogenase is a huge enzyme complex, made of two proteins - reductase (Fe protein as it contains iron centres) and nitrogenase (MoFe protein as it contains a molybdenum iron cluster which is important)

What are the reactions of nitrogenase?

• N2 + 6e- + 6H+ --> 2NH3

• N2 + 8e- + 8H+ --> 2NH3 + H2

What do the reactions of nitrogenase require?

• This requires electrons with a very negative redox potential, these are from ferredoxin (electron carrier in photosynthesis). Ferredoxin redox potential is very negative, cannot use NADH for this nitrogen fixing process. The process of producing the negative redox potential electrons requires ATP, but the main purpose of ATP is to reduce the activation energy - makes the reaction plausible and workable at room temperature (not make it overall more thermodynamically favourable)

Describe the reactions of nitrogenase

• Electrons in ferredoxin bind to the iron protein, this iron protein hydrolyses ATP and this allows it to transfer electrons to the nitrogenase protein, in a sequential manner, slowly reducing the nitrogen gas form nitrogen to ammonia. This is energetically costly (need to use ATP and generate very negative electrons).

Describe inhibition of nitrogenase and how this can be overcome

• Metal centres are oxygen sensitive, so nitrogenase will not work under oxygen conditions overcome by leghaemoglobin (legumes), binds with high affinity which allows them to lower the oxygen concentration in the root nodule

• Ideally we would use oxygen as an electron acceptor as you can get more energy out of the substrates (larger redox potential difference)

• Different equivalent to complex 4 in their ETC, this has a very high affinity for oxygen, so can use oxygen at very low concentrations as an electron acceptor, this allows them to overcome the poisoning of the nitrogenase but still being able to produce energy