Nitrogenase

• two steps of all amino acid biosynthesis

  1. nitrogen fixation by enzyme nitrogenase
  2. reduction of alpha-ketoglutarate to glutamate by glutamate dehydrogenase
• Main source of biological nitrogen?

  Nitrogen fixation from nitrogenase in bacteria!
• nitrogen fixation = atmospheric N2 is reduced to ammonia NH3, requires enzyme nitrogenase
• this reaction is spontaneous and exothermic, but very slow

  
• nitrogenase requires 16 ATP to help speed up reaction; also uses 16 water and 8 electrons
• nitrogenase components:

  • reductase (Fe protein)
  • nitrogenase (MoFe protein)
• electron movement in Fe protein: ferredoxin (Fd) -> Fe protein (specifically [4Fe-4S] cluster) -> out to MoFe protein
• Fe protein is a homodimer, where each dimer has its own [4Fe-4S] cluster that can pick up an electron from Fd
• Fe protein uses 2 ATP per electron, where ATP hydrolysis is used to trigger a conformational change
• the iron-sulfur clusters in Fe protein are anchored at the iron atoms, which form tetrahedral structures by bonding to cysteines of the protein
• MoFe protein is a heterotetramer, or like a dimer of the alpha-beta dimers

  
• label some parts of Mofe protein in nitrogenase

  A) P cluster

  B) FeMoco

  C) homocitrate

  D) Mo

  
• each alpha-beta of MoFe protein has its own active site, with its own two metal clusters -- P cluster and FeMoco
• P cluster = two [4Fe-4S] clusters that share a central sulfur atom; in MoFe protein, it will pick up one e- at a time from Fe protein to deliver to FeMoco
• FeMoco (FeMo cofactor) = a [Mo-7Fe-9S-C] cluster w/ homocitrate ligand; in MoFe protein, most likely the active site where nitrogen actually gets reduced or fixed
• FeMoco is bound (held in place in the enzyme) by two alpha subunits

  A) Cys

  B) His

  
• One hypothesis for FeMoco mechanism = N2 displaces the belt sulfurs in the Fe-S-C cluster
• leghemoglobin

  • similar to hemoglobin, but only one chain and one heme
  • chain made in the plant, heme made in the bacteria
  • binds O2 in root nodules and acts as an O2 buffer to balance out O2 for both (anaerobic) nitrogenase activity and aerobic respiration