1. Nitrogen Fixation Our goal is to learn how N2 an inert gas, becomes part of the structure of organic molecules Secondly, to study the function of nitrogen compounds in plants and bacteria To study the nitrogenase complex and learn its secrets for fixing nitrogen

2. 450oC (Super heated Steam) How Powerful is Nitrogen Gas? Haber Process 300 atm N2(g) + 3H2(g) 2NH3(g) + 92 kJ

3. N N N N Triple bond Why is Nitrogen so Hard to Split? 945 kJ per mole (C-C Bond is 347 kJ per mole) + 2e + 2H+ HN=NH HN=NH + 2e + 2H+ H2N-NH2 H2N-NH2 + 2e + 2H+ 2NH3

4. Valence State is a Key to the Chemistry of N +5 HNO3 Nitrates +4 NO2(g) OXIDATION Nitrites HNO2 +3 +2 NO Nitric oxide +1 N2O(g) Diatomic 0 N2 -1 HN=NH REDUCTION -2 H2N-NH2 -3 NH3 Ammonia

5. Nitrogen Compounds in Plants and Bacteria Nitrite Reduction Return nitrogen compounds to N2 Nitrate Reduction Denitrification NO3 NO2 N2(g) NH3 Energy Energy Nitrification (Soil bacteria) N2 fixation (reduction) Plants produce NH3 by reducing nitrates and nitrites Soil bacteria derive energy by oxidizing NH3

6. Nitrogen Metabolism by Species Bacteria Plants Animals Yes No No Nitrogen fixation Yes Yes No Nitrate Reduction Nitrite Reduction Yes Yes No Nitrification Yes No No Denitrification No Yes No NH3 Assimilation Yes Yes Yes Amino Acid Synthesis Yes Yes Yes

7. Remarkable Nitrogenase Biological Reaction N2 + 8H+ + 8e- + 16ATP + 16H2O 2NH3 + 16ADP + 16Pi + H2 + 16H+ Nitrogenase Complex (nif gene cluster) Nitrogenase (Mo, Fe protein) Nitrogenase Reductase (Fe protein)

8. Nitrogenase Dialogue Where do we find Nitrogenase? Nitrogenase is present in certain bacterial species referred to a diazotrophs. The legumes (peas, beans, alfalfa, etc.) have root nodules infested with the nitrogen fixing bacteria, Rhizobium. Free living bacteria such as azobacter vinelandii can also fix nitrogen. What is meant by symbiosis in nitrogen fixation? Symbiosis means living together for mutual benefit. The bacteria provide the plant with NH3 and the plant prevents oxygen from interfering with the fixation reaction.

9. What interferes with nitrogen fixation? Oxygen is the major factor. Nitrogen fixation can take place only in the total absence of O2 How does a plant overcome oxygen interference? By synthesizing a heme protein, leghemoglobin, which like other hemoglobins, has a high affinity for binding oxygen….what is called “sequestering” the oxygen. Why such as funny name? The name gives the origin of the protein. It is made in legumes, hence leghemoglobin. The heme group of the hemoglobin is made in the bacteria.

10. Why are there two components in the nitrogenase complex One component holds the N2 molecules, the other passes electrons. Nitrogenase is a two-step energy machine in which electrons are boosted to a higher reducing potential (Nitrogenase reductase or Fe-protein) and then passed to the N2 bound to the MoFe-protein SHOW ME

11. Electron Transfer “Pump” Dinitrogenase M o A c t i v a t e d 0 . 4 0 M o - H - F d - e R M o - H N = N H - 0 . 2 9 F d R e H - M o - H A T P A D P H H 2 Dinitrogenase Reductase H - M o = N = N - F d - e R M o - F d R e A T P A D P H - M o = N - N H 2 e + H+ - F d - e R N H 3 e + H+ - F d R e A T P A D P N H N M o = 3 e + H+ Nitrogenase MoFe-Protein Reductase Fe-Protein

12. CO2 ATP NH3 Carbamoyl phosphate Glutamate Glutamine Asparagine None Other amino acids Purine nucleotides, Cytidine nucleotides Amino sugars, Tryptophan, Histidine Pyrimidine nucleotides Arginine Urea The only inorganic nitrogen source for mammals is NH3 3 Gateways to Biological Molecules a-Ketoglutarate Aspartate Glutamate Of the 3, the most versatile is glutamine

13. Rapid Quiz • What key biocompound allows a bacterium, a fungus, or a hippopotamus to convert carbohydrates and amino acids into fat? • Based on your answer, show how the carbons in the amino acid alanine and glucose become carbons in cholesterol • Outline a path to show how the carbons in phenylalanine become the carbons in stearic acid and glucose.

14. Answer Aminoacids Carbohydrates Acetyl-CoA Fatty acids Cholesterol Acylglycerolphosphates Triacylglycerols Sphingolipids

15. Alanine Glucose Stearate Pyruvate Acetyl-CoA HMG-CoA Mevalonate Isopentenyl-PPi Cholesterol

16. Phenylalanine Fumarate Acetoacetate Tyrosine L-malate Acetyl-CoA p-hydroxyphenylpyruvate Malonyl-CoA OAA Homogentisate PEP Palmitate Maleyl-acetoacetate Gluconeogenesis Stearate Fumaryl-acetoacetate Glucose