1. February 12, 2002 Chapter 26 Nitrogen Acquisition
Biochemistry 432/832
February 12, 2002
Chapter 26
Nitrogen Acquisition

2. Announcements: -

3. Outline 26.1 The Two Major Pathways of N Acquisition
26.2 The Fate of Ammonium
26.3 Glutamine Synthetase
26.4 Amino Acid Biosynthesis
26.5 Metabolic Degradation of Amino Acids

4. Major Pathways for N Acquisition
All biological compounds contain N in a reduced form
The principal inorganic forms of N are in an oxidized state
Thus, N acquisition must involve reduction of the oxidized forms (N2 and NO3-) to NH4+
Nearly all of this is in microorganisms and green plants
Animals gain N through diet.

5. The nitrogen cycle

6. Overview of N Acquisition
Nitrogen assimilation and nitrogen fixation
Nitrate assimilation occurs in two steps: 2e- reduction of nitrate to nitrite and 6e- reduction of nitrite to ammonium
Nitrate assimilation accounts for 99% of N acquisition by the biosphere
Nitrogen fixation involves reduction of N2 in prokaryotes by nitrogenase

7. Electrons are transferred from NADH to nitrate
Nitrate Assimilation
Electrons are transferred from NADH to nitrate
Pathway involves -SH of enzyme, FAD, cytochrome b and MoCo - all protein-bound
Nitrate reductases are big kDa
MoCo required both for reductase activity and for assembly of enzyme subunits to active dimer

8. Novel prosthetic groups used in N acquisition
Molybdopterin
Siroheme

9. Mo-containing enzymes
Mo is the heaviest element used by eukaryotes
Two classes of molybdoenzymes
1) Molybdopterin-dependent enzymes
Nitrate reductase
Formate dehydrogenase
Aldehyde oxidase
Xanthine dehydrogenase
Sulfate oxidase
2) Nitrogenase
Molybdopterin

10. Nitrite Reductase
Light drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitrite
Nitrite is reduced to ammonium while still bound to siroheme
In higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolic

12. Enzymology of N fixation
Only occurs in certain prokaryotes
Rhizobia fix nitrogen in symbiotic association with plants
Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates
All nitrogen fixing systems are very similar
They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits reaction and NH4+ inhibit expression of nif genes)

13. Two protein components: nitrogenase reductase and nitrogenase
Nitrogenase Complex
Two protein components: nitrogenase reductase and nitrogenase
Nitrogenase reductase is a 60 kDa homodimer with a single 4Fe-4S cluster
Very oxygen-sensitive
Binds MgATP
4ATP required per pair of electrons transferred
Reduction of N2 to 2NH3 + H2 requires 4 pairs of electrons, so 16 ATP are consumed per N2

14. Why should nitrogenase need ATP???
N2 reduction to ammonia is thermodynamically favorable
However, the activation barrier for breaking the N-N triple bond is enormous
16 ATP provide the needed activation energy

15. To break the triple bond, energy input in necessary

16. Nitrogenase A 220 kDa heterotetramer
Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc.)
Four 4Fe-4S clusters plus two FeMoCo, an iron-molybdenum cofactor
Nitrogenase is slow - 12 e- pairs per second, i.e., only three molecules of N2 per second

17. Structures of two types of metal clusters in nitrogenase:
The P-cluster
FeMoCo

18. The nitrogenase reaction
Accumulation of electrons

19. Complex between nitrogenase reductase and nitrogenase

20. Regulation of nitrogen fixation
ADP inhibits
NH4+ represses expression
ADP-ribosylation inhibits

21. Three major reactions in all cells
The Fate of Ammonium
Three major reactions in all cells
Carbamoyl-phosphate synthetase
two ATP required - one to activate bicarbonate, one to phosphorylate carbamate
Glutamate dehydrogenase
reductive amination of alpha-ketoglutarate to form glutamate
Glutamine synthetase
ATP-dependent amidation of gamma-carboxyl of glutamate to glutamine

22. The glutamate dehydrogenase reaction

23. The glutamine synthetase reaction

24. Ammonium Assimilation
Two principal pathways
Principal route: GDH/GS in organisms rich in N
both steps assimilate N
Secondary route: GS/GOGAT in organisms confronting N limitation
GOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferase

25. The glutamate dehydrogenase/glutamine synthase pathway
Two N fixing steps - one inefficient
One each

26. The glutamate synthase reaction

27. The glutamine synthase/GOGAT pathway
One N fixing step - inefficient but expensive
One NADPH
Two ATP

28. Glutamine Synthetase A Case Study in Regulation
GS in E. coli is regulated in three ways:
Feedback inhibition
Covalent modification (interconverts between inactive and active forms)
Regulation of gene expression and protein synthesis - - control the amount of GS in cells

29. The glutamine synthetase reaction

30. Glutamine synthetase structure
stack of two hexagons

31. Allosteric Regulation of Glutamine Synthetase
Nine different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P and glucosamine-6-P
Gly, Ala, Ser are indicators of amino acid metabolism in cells
Other six are end products of biochemical pathways
This effectively controls glutamine’s contributions to metabolism

32. Allosteric regulation of glutamine synthase activity by feedback inhibition

33. Covalent Modification of Glutamine Synthetase
Each subunit is adenylylated at Tyr-397
Adenylylation inactivates GS
Adenylyl transferase catalyzes both the adenylylation and deadenylylation
PII (regulatory protein) controls both activities
AT:PIIA catalyzes adenylylation
AT:PIID catalyzes deadenylylation
-ketoglutarate and Gln also affect

34. Covalent modification of glutamine synthase - adenylylation of Tyr397