Nitrogen Metabolism

Question 1

Nitrogen in the atmosphere

Answer 1

• most abundant element in air (80%)

- occurs as dinitrogen

Question 2

Nitrogen fixers

Answer 2

• convert N2 to ammonia

Question 3

Nitrogen fixation

Answer 3

• conversion of N2 into NH3-
• the “fixed” nitrogen is immediately incorporated into organic compounds
• only prokaryotes fix nitrogen
• NO nitrogen fixing plants or animals.

Question 4

Ammonification

Answer 4

• release of ammonia into the environment from N-containing organic compounds

Question 5

Nitrification

Answer 5

• oxidation of ammonia by aerobic bacteria

Question 6

Denitrification

Answer 6

• reduction of nitrate to N2 by anaerobic respiration

Question 7

biological Nitrogen fixation reaction

Answer 7

• N2 + 8H+ + 8e- + 16-32 ATP -> 2 NH3+ H2 + 16-32 (ADP + Pi)

Question 8

Non biological nitrogen fixation

Answer 8

• N2 + 3H2 -> 2 NH3
• Haber-Bosch reaction
• accounts for 1/5 of nitrogen fixed per year

Question 9

Why is nitrogen fixation so expensive?

Answer 9

• N2 is joined by a very stable triple bond that is difficult to break.

Question 10

Who fixes nitrogen?

Answer 10

• Free living heterotrophs - Azotobacter and Pseudomonas
• Phototrophs - Anabaena, Nostoc, Rhodospirrilum, Rhodobacter
• Plant symbiotic bacteria
  
  • Rhizobium - leguminous plant
  • Frankia - angiosperms
  • Azospirillum - grass, maize

Question 11

Nitrogenase

Answer 11

• carries out nitrogen fixation

Question 12

nitrogen is reduced at

Answer 12

• molybdenum-iron cofactor

- reducing power stored in P-cluster - stores electrons

Question 13

Nitrogenase reguluation

Answer 13

• you must make 26 proteins to support N2 fixation
  
  • protein synthesis is expensive
• it takes 16-32 ATP + NADH per N2 fixed
• regulated at transcriptional level and post-translational level

Question 14

Transcriptional Regulation

Answer 14

• NtrB and NtrC two component regulatory system
• NtrB is the sensor - senses N status of the cell
• NtrC is the response regulate - activates transcription of NifA, NifL
• NifA - positive regulator
• NifL - negative regulator

Question 15

nitrogen status measured by

Answer 15

• measured to the ratio of alpha-ketoglutarate/glutamate via PII

Question 16

Transcriptional regulation process

Answer 16

• nitrogenase extremely sensitive to O2. Only turn on if O2 is low
• in presence of high levels of O2, NifL will bind to NifA and not allow it to turn on transcription of nitrogenase operon
• If O2 levels are low, NifL will not bind NifA, and it can activate transcription

Question 17

Post translational regulation

Answer 17

• the cell shuts off nitrogenase whenever ammonia feels increase by modifying an arginine residue in the active site of the iron protein.
• done by adding an ADP ribose via DRAT.
• when ammonia levels drop, the ADP ribose removed by DRAG.

Question 18

Nitrogenase and O2

Answer 18

• nitrogenase is rapidly inactivated by exposure to O2
• The FeMoCo center binds to O2 tightly, and the entire enzyme must be degraded and made again
• Many N2 fixers have developed mechanisms to protect nitrogenase from O2

Question 19

Azotobacter vinelandii

Answer 19

• obligate aerobe, but can fix N2
• to protect nitrogenase from inactivation use
  
  • respiratory protection
  • conformational protection

Question 20

Respiratory protection

Answer 20

• Azotobacter keeps its cytoplasm at very low O2 concentration through its ETC
• At high O2 levels, it will turn on an alternative terminal oxidase, cytochrome bd, that has a high affinity for O2 and a very rapid rate of O2 reduction
• but it’s not a proton pump, so less energy available

Question 21

Conformational protection

Answer 21

• if O2 is too high, FeSII will bind to nitrogenase and protect the FeMO cluster from O2 inactivation
• When FeSII is bound to the protein, N2 also does not have access and nitrogen fixation stops
• When O2 drops, FeSII dissociates and nitrogen fixation proceeds.

Question 22

Cyanobacteria

Answer 22

• cannot fix nitrogen and photosynthesize at the same time because O2 will damage nitrogenase
• when these bacteria need to fix nitrogen, they do so in a heterocyst and share nitrogen with neighbors who share their fixed CO2
• no photosynthesis occurs in a heterocyst so nitrogenase is not inhibited.

Question 23

symbiotic bacteria

Answer 23

• nitrogen fixing bacteria live in symbiosis with plants

- environment inside the nodule kept at low O2 by leghemoglobin

Question 24

lock and key mechanism

Answer 24

• only bacteria that secrete the correct polysaccharide “key” are able to colonize each host.
• plant secretes flavonoids that stimulate bacteria to start producing Nod factors
• bacteria make polysaccharide which induces root hair curling and allows the bacteria to invade into the cortex cells.
• will invade cell and divide rapidly producing a nodule on the root.

Question 25

Ammoniafication

Answer 25

- mineralization - once N2 is fixed to ammonia, it is almost immediately incorporated into amino acids - when an organism is growing on high nitrogen:carbon ratio diet - will excrete ammonia

Question 26

nitrification

Answer 26

- aerobic process | - bacteria of the genus nitrosomas and nitrobacter oxidize ammonia to nitrite and nitrate

Question 27

Nitrosomas

Answer 27

- gets energy from oxidation of ammonia to nitrite - does in two steps - first step produces hydroxylamine - then oxidizes to nitrite

Question 28

nitrobacter

Answer 28

- uses nitrite as energy source to produce nitrate

Question 29

Denitfification

Answer 29

- nitrate (NO3) to Dinitrogen (N2) - anaerobic process - nitrate - nitrite - nitric oxide - nitrous oxide - dinitrogen