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:

NN + 2H+ + 2e à HN=NH  (Diimine)

HN=NH + 2H+ + 2e à H2NNH2 (Hydrazine)

H2NNH2 + 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 PH, 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