NITROGEN FIXATION

· N₂ is quite stable (The N≡N has a bond energy of 945 kJ mol⁻¹, versus 351 kJ mol⁻¹ for C−O bond).

· Requires a substantial amount of energy to fix N₂ into NH₃.

· Can be done by a few strains of bacteria – diazotrophs (such as certain marine cyanobacteria and bacteria that is symbiotic in the roots of legumes).

· Diazotrophs contain “nitrogenase” that catalyzes:

N₂ + 8H⁺ + 8e⁻ + 16 ATP + 16 H₂O à 2 NH₃ + H₂ + 16 ADP + 16 Pi

NITROGENASE

A multi-subunit complex of two proteins (see Fig.20-41)

1. A Fe-protein homodimer g₂ containing one [4Fe-4S] cluster and 2 ATP binding sites.

2. A MoFe-protein α₂β₂ hetero-tetramer containing Mo-Fe cofactor and a P-cluster (consisting of two iron-sulfur clusters).

Mechanism of Nitrogenase

· The required electrons are derived from photosynthesis or oxidative electron transport.

· See Fig. 20-43 for electron transfer from Fe-protein to MoFe-protein to N₂, and ATP utilization.

· 2 ATP are utilized for the transfer of one electron.

· 2 ATP bind to reduced Fe-protein and are hydrolyzed as the electron is transferred to MoFe-protein.

· The ATP hydrolysis causes a conformational change of Fe-protein, altering its E°’ from -0.29 to -0.40 V. This enables the subsequent reduction of N₂ to 2NH₃ (E°’ = -0.34 V for N₂ + 6H⁺ + 6e⁻ ⇌ 2 NH₃)

· This occurs in three steps of 2e-reduction:

N≡N + 2H⁺ + 2e⁻ à H−N=N−H (Diimine)

H−N=N−H + 2H⁺ + 2e⁻ à H₂N−NH₂ (Hydrazine)

H₂N−NH₂ + 2H⁺ + 2e⁻ à 2 NH₃

· The step-wise reduction of N₂ occurs on the MoFe-protein.

· However, for every N₂ reduced to 2 NH₃, at least one additional N₂ goes through a 2e-futile cycle via diimine:

N₂ + 2H⁺ + 2e⁻ à HN=NH

HN=NH + H₂ à N₂ + 2 H₂

· Therefore, the overall reaction for the net formation of 2 NH₃ from one N₂ is:

N₂ + 8H⁺ + 8e⁻ + 16 ATP + 16 H₂O à 2 NH₃ + H₂ + 16 ADP + 16 Pi

WHAT HAPPENS TO FIXED NH₃?

1. Key Role of Glutamine Synthetase

· Glutamine synthetase catalyzes the formation of glutamine from ammonia, ATP, and glutamate.

Glutamate + ATP + NH₄⁺ à Glutamine + ADP + Pi

· Glutamine is a major amino group donor and a storage form of NH₃.

· Glutamine synthetase is a key to the control of nitrogen metabolism, and is sensitive to activity regulation.

· Mammalian enzyme is activated by α–ketoglutarate, the product of glutamate oxidative deamination (see below). The activation will prevent the accumulation of NH₃.

· Bacterial enzyme consists of 12 identical subunits arranged in two layers of hexamer.

· Sensitive to allosteric inhibitions by (1) histidine, tryptophan, carbamoyl phosphate, AMP, and CTP (all end products derived from glutamine) and (2) alanine, serine, and glycine (markers for cellular nitrogen level).

· E. coil glutamine synthetase is sensitive to covalent modification by adenylylation of a tyrosine residue involving a regulatory protein P_H, adenylyltransferase, uridylyltransferase, and uridylyl-removing enzyme. (See Fig. 20-29).

2. Other enzymatic reactions

Including mitochondrial glutamate dehydrogenase (catalyzes a reversible reaction) and carbamoyl phosphate synthetase I, etc.

Glu + NAD⁺ (or NADP⁺) + H₂O ⇌ NH₄⁺ + a-ketoglutarate + NAD(P)H + H⁺

2ATP + HCO₃⁻ + NH₃ à Carbamoyl phosdphate + 2ADP + Pi

NITROGEN CYCLE

Describes the interconversion of nitrogen in the biosphere.

Ammonification

By plants, fungi, and many bacteria.

Nitrate Reductase: NO₃⁻ + 2 H⁺ + 2 e⁻ à NO₂⁻ + H₂O

Nitrite Reductase: NO₂⁻ + 7 H⁺ + 6 e⁻ à NH₃ + 2 H₂O

Anammox

In some newly discovered strains of anaerobic bacteria, which contain anammoxosome.

NH₄⁺ + NO₂⁻ à N₂ + 2 H₂O