1. Nitrogen cycles Nitrate/nitrite Reductase Ammonia monooxygenase Hydroxylamine oxidoreductase

2. N 2 + 3H 2 ---> 2NH 3 ∆G’º = -33.5 kJ/mol However, N-N triple bond is a significant kinetic barrier Bond energy = 930 kJ/mol N 2 + 10 H + + 8 e - + 16 ATP ---> 2 NH 4 + + 16 ADP + 16 P i + H 2 Nitrogen is fixed by anaerobic bacteria 0 -3

4. FeMo Cofactor

5. Proposed mechanism for dinitrogen reduction

6. Nitrate:ferredoxin reductase

7. Dissimilatory/Respiratory Nitrite Reductase Siroheme and ferredoxin dependent looks and behaves a lot like cytochrome c oxidase NO 2 - ----> NO ----> NH 2 OH ----> NH 4 + Allows anaerobic organisms to respire electrons onto nitrite instead of oxygen +3 +1 -3

8. Dissimilatory Nitrite Reductase NO 2 - ----> NO Produces NO and N 2 O - potent greenhouse gasses +3 +2 NO ---> N 2 O +2 +1 Nitrite reductase, multicopper enzyme Nitric oxide reductase, heme dependent N 2 O ---> N 2 +1 0 Nitrous oxide reductase, multicopper enzyme

9. Biological Nitrification NH 3 ---> NO 2 - ---> NO 3 - -3 +3+5 NH 3 + O 2 + 2 e - + 2 H + ---> NH 2 OH + H 2 O Ammonia monooxygenase (related to methane monooxygenase) NO 2 - ---> NO 3 - is non-enzymatic

10. NH 2 OH + H 2 O ---> NO 2 - + 5 H+ + 4 e - Hydroxylamine oxidoreductase (heme dependent) 2 of these electrons are transferred to cytochrome bc 1 Ultimately they land on O 2 and generate proton gradient 2 electrons are returned to AMO to complete the cycle

12. Assimilation of ammonia Use glutamate and glutamine as reservoirs

13. Glutamine synthase glutamate + NH 4 + + ATP ---> glutamine + ADP + P i + H + Glutamate synthase glutamine +  KG + NADPH + H + ---> 2glutamate + NADP + Net reaction  KG + NADPH + NH 4 + + ATP ---> glutamate + NADP + + H 2 O + ADP + P i Need catalytic amounts of glutamate! There has to be another way!

14. Glutamine synthase

15. Glutamate synthase

17. What about the reverse reaction of glutamate dehydrogenase? glutamate + NADP + + H 2 O --->  KG + NH 4 + + NADPH Normally this reaction favors the right

19. Glutamine synthetase is a control point for growth. Many end products allosterically regulate its activity

23. Amino transferases

30. Carbamoylphosphate synthase: step 1 Carbonic phosphoric anhydride Bicarbonate

31. Carbamoylphosphate synthase: step 2 Carbonic phosphoric anhydride Carbamoylphosphate Carbamate

32. Ornithine transcarbamoylase + P i Ornithine Citrulline These are  -amino acids

33. Argininosuccinate synthase: step 1 Citrulline Citrullyl-AMP

34. Argininosuccinate synthase: step 2 Citrullyl-AMP Argininosuccinate

35. Argininosuccinate lyase Argininosuccinate Arginine Fumarate

36. Arginase

38. The aspartate/malate shuttle

39. In respiration asp/mal shuttle Moves NADH into the mitochondrion with net movement of one H + in but also moves NH 3 out! For every asp out a mal goes in and NH 3 is moved out to cytoplasm

40. Sulfur Assimilation S 2- ---> S ---> SO 3 2- ---> SO 4 2- -2 0+4 +6

41. Sulfide oxidation by photosynthetic microbes e - from sulfide

42. S 2- + cyt c 553(ox) ---> S + cyt c 553(red) Sulfide dehydrogenase Sulfide:quinone oxidoreductase S 2- + UQ (ox) ---> S + UQH 2(red)

43. S 2- ---> S ---> SO 4 2- In photosynthetic green sulfur bacteria this oxidation is the driving force for the rTCA cycle Elemental sulfur globules are temporarily deposited outside the cell.

44. Sulfur oxidation Sulfur oxygenase reductase (SOR) Mononuclear non-heme iron enzyme SOR S + O 2 ----> HSO 3 - + H + Non-enzymatic disproportionation 3S + 3H 2 O ----> HSO 3 - + 2HS - + 3H + Sum 4S + O 2 + 4H 2 O ----> 2HSO 3 - + 2HS - + 4H + Non-enzymatic reaction with more S S + HSO 3 - ----> S 2 O 3 2- + H +

45. Sulfite oxidase converts sulfite to sulfate

46. Thiosulfate:quinone oxidoreductase Tetrathionate S 4 O 6 2- + H 2 O ---> S 3 O 3 2- + SO 4 2- + 2H + Tetrathionate hydrolase S 3 O 3 2- ---> S 2 O 3 2- + S Spontaneous disproportionation 2S 2 O 3 2- S 4 O 6 2-

47. How to assimilate sulfate? typo

48. NADPH reduces the disulfide PAPS Reductase

49. Sulfate/ite respiring organisms use dissimilatory sulfate/ite reductase This is a respiratory enzyme that couples sulfate/ite reduction with NADPH oxidation via ferredoxin related to cytochrome c oxidase Catalyzes the reduction of sulfite to sulfide

50. Sulfide is assimilated by incorporation into cysteine

51. Acetylserine lyase

53. Steegborn, C. et al. J. Biol. Chem. 1999;274:12675-12684 Overview of the transsulfuration reactions

54. Cystathionine-  -synthase

57. Cystathionine-  -lyase Homocysteine

59. Methionine synthase

60. In mammals cysteine is made from methionine

62. SAM-methyltransferase/S-adenosylhomocysteine hydrolase

64. Cystathionine-  -synthase

65. Cystathionine-  -lyase

67. Cysteine desulfurylase: looks like serine dehydratase