Fritz Haber 1868 - 1934

• 1918 Nobel prize “synthesized ammonia from its elements”

Annual Global N-fixation

Type of fixation N2 fixed ( x 106 metric tons year-1)

Non-biological Industrial 50 Combustion 20 Lightning 10Total non-biological 80

Biological Agricultural land 90 Forest and non-agricultural land 50 Sea 35Total biological 175

Data from various sources, compiled by DF Bezdicek & AC Kennedy, in Microorganisms in Action (eds. JM Lynch & JE Hobbie). Blackwell Scientific 1998.

Possess homologs to NifH and Nif D but not known to fix N2

Include taxa that fix N2

Do not include taxa that fix N2

Raymond et al 2004 Mol. Biol. Evol. 21:541-554

Nitrogenase enzyme complex

MoFe protein• Dinitrogenase• Tetramer of two different dimers

2 2 heterodimer• = NifD, = NifK

• Contains– Two MoFeS centers (active site for N2

reduction)

– Four Fe4S4 centers

• Combines N2 and H+ to reduce N2 to NH3

Nitrogenase enzyme complex

From: Catalina Achimhttp://www.chem.cmu.edu/groups/achim/research/magneto.html

Fe protein

Fe protein

MoFe protein NH3

N2

190 Å

65 Å

e-

Nitrogenase enzyme complex

Fe protein

• Dinitrogenase reductase

• Dimer of two identical subunits2 dimer

= NifH

• Contains single Fe4S4 center

• Accepts reducing power (electrons) and reduces MoFe protein (dinitrogenase)

Nitrogenase enzyme complex

From: Catalina Achimhttp://www.chem.cmu.edu/groups/achim/research/magneto.html

Fe protein

Fe protein

MoFe protein NH3

N2

190 Å

65 Å

e-

Nitrogenase enzyme complex

Name Formula Major products

Proton H+ H2

Dinitrogen NN NH3 + H2

Nitrous oxide NN+-O- N2 + H2O

Azide [NN+-N]- N2 + NH3 + N2H4

Acetylene HCCH H2C=CH2

Cyanide [CN]- CH4 + NH3

Carbon monoxide CO CO (binds to N-binding site but is notreduced. Blocks rxnwith other substrates)

Versatility of nitrogenase complex

Vesicles are the site of N2-fixation in Frankia. Nitrogenase reduces nitrogen using electrons and ATP from compounds donated by the plant (succinate is only one possibility). The product ammonia is most likely excreted from the vesicle where plant enzymes (GS) convert it to organic nitrogen (glutamine). (D. Benson)

Ferredoxinreduced

Ferredoxinoxidized

e-

e-

Feox.

Fered.

e-

2 ADP

2 ATP

MoFered.

MoFeox.

N2, 8H+

2 NH3,H2

e- e-

e-

2 Pi

Nitrogenase enzyme complex

From: Catalina Achimhttp://www.chem.cmu.edu/groups/achim/research/magneto.html

Fe protein

Fe protein

MoFe protein NH3

N2

190 Å

65 Å

e-

Nitrogenase enzyme complex

Groups I - IIIpossess functional nitrogenases

Raymond et al 2004 Mol. Biol. Evol. 21:541-554

Tree of NifH and NifD homologs

Two proposed nif gene vs. spp. trees

(a) Last common ancestor (LCA) hypothesis

(b) Methanogen origin hypothesis