The Case for Nitrogenase

         

           The abundance of dinitrogen (N2) in the earth's atmosphere and in other planetary atmospheres displays just how energetically stable the N≡N bond is. Having a bond enthalpy of nearly 950 kJ/mol ensures dinitrogen is the strongest existing bond, and keeps dinitrogen significantly nonreactive. Industrially, nitrogen is converted to ammonia in the Haber-Bosch process, requiring a catalyst, temperatures in excess of 200 C and at pressures in excess of 150 atm. Fertilizer from ammonia produced via the harsh conditions of the Haber-Bosch process accounts for over half the world's food production.

           But yet, nature, utilizing nitrogenase, performs the very same reaction at ambient conditions. The prevalence of nitrogen atoms in proteins, DNA, RNA, and other bio-molecules displays its biological importance. Nitrogen is, clearly, one of the fundamental building blocks of life and, staggeringly, ALL of it is fixed, or made usable, by a single enzyme: nitrogenase. This fixed nitrogen is then incorporated into every single biological macromolecule. The stream of life truly begins here, at nitrogenase!

Fig. 1: The heterotetrameric structure of nitrogenase, pictured with homodimeric Fe protein subunits.
(PDB code: 1N2C. Figure generated using Pymol.)

Reaction

          Nitrogenase catalyzes the 8 e- reduction of gaseous dinitrogen, yielding ammonia and hydrogen gas. This reaction takes place in the root-nodules of leguminous plants. To overcome the huge activation barrier (~420 kJ/mol), nitrogenase requires the energetic impetus from 16 ATP molecules, added via the Fe protein subunits, and stability provided via a molybdenum atom. Additionally, it incorporates FeS cofactors to perform the reaction... not just one cluster, nitrogenase contains three FeS clusters.

Overall Reaction:      N2 + 16 ATP + 8 e- + 8 H+ ⇒ 2 NH3 + H2 + 16 ADP   (ΔG_rxn = - 93 kJ/mol)

Structure & Mechanism

          Despite the continuing effort of numerous scientists, not much light has been shed on the mechanism of nitrogenase action. This is due, significantly, to the difficulty in trapping the highly reactive N-intermediates of the reaction. As a result, nitrogenase has been dubbed 'The Everest of Enzymes', displaying both the complexity of the mechanism and the enzyme's legendary status in the scientific community. To find out more, read paper 3 of the previous blog entry.

                   

          Yet, there is far more to be said about what is known of nitrogenase structure. To be concise, I will restrict myself to 3 key features of the enzyme: (1) Unique FeS clusters & cofactors, (2) electron transport in the enzyme, and finally  (3) mechanism of preventing oxidation.

Fig. 2: Cofactor ensemble of nitrogenase(3)

(1) Unique FeS Clusters: As mentioned earlier, nitrogenase contains 3 FeS clusters. The first of these, an Fe₄S₄ cluster, is impressive on its own merit. The second cluster, termed the P cluster, is an outrageous Fe₈S₇ cluster (one of the biggest naturally occuring FeS cofactors). The uniqueness of nitrogenase does not end there. The next cofactor, an FeMo-co cluster, trumps the lot. It is thought the reaction occurs on the face of this cluster, which incorporates a Molybdenum(!) metal center and a homocitrate molecule just to stabilize the molybdenum (see fig. (b) above).

(2) Electron Transport in Protein: The association of nucleotide-bound Fe-protein subunits to the MoFe protein subunit leads to the release of an electron from ATP towards the first Fe₄S₄ cluster, which shuttles the electron towards the larger P-cluster. The P-cluster gains further electrons when another Fe-protein binds, eventually passing them on towards the FeMo-co reactive center, which, by the way, has a central carbon atom with 6 bonds to it. Note that this entire process goes on simultaneously on the other half of the protein, making nitrogenase that much more efficient.

(3) Prevention of Oxidation: For a successful dinitrogen reduction to occur, it is imperative that any, and all oxidizing agents are kept well clear of the catalytic site. Rather conveniently, the binding of Fe-proteins subunits to the Fe-Mo protein, using a 'β-grasp' structure, seals off the Fe-Mo protein from oxidation. Even if oxidation were to occur, it would be at the interface of the smaller FeS cluster, thereby protecting the Fe-Mo-co cluster and facilitating further enzymatic reactions to occur.  

Fig. 3: Protein subunit interface.
(PDB code: 1N2C. Figure generated using Pymol.)

Why Nitrogenase Deserves to Win

             Simply put, nitrogenase performs the most biologically difficult reactions there is: a reduction of dinitrogen. It so happens, that this reaction is absolutely vital for every living organism. In order to perform this reaction, nitrogenase employs a number of novel structural features, such as two FeS clusters, a molybdenum metal-center, and a carbide, with 6 bonds to carbon. In unison, these unusual features enable nitrogenase to perform some simply phenomenal chemistry.

       

             Should you choose not to vote for it, consider the fate of every protein, DNA & RNA molecule in your body without nitrogen. None of it would polymerize and any bonds that formed would be totally insufficient to support life... all you would be is a pile of oxidized carbon.

References:

1. Science , New Series, 2002, 297, 5587, pp. 1696-1700
2. Rees, D.C. and Howard, J.B.; Current Opinion in Chemical Biology, 2000, 4, pp. 559-566
3. Sgrignani, J.; Franco, D.and Magistrato, A.; Molecules, 2011, 16, pp. 442-465
4. Kim, J. and Rees, D.C.; Biochemistry, 1994 33, pp. 389-397
5. Hoffman. B.M.; Dean, D.R.; Seefeldt, L.C.; Acc. Chem. Res. 2009, 42, pp. 609-619
6. Burgess, B.K. and Lowe, D.J.; Chem. Rev. 1996, 96, pp. 2983-3011
7. Evans, D.J. and Pickett, C.J.; Chem. Soc. Rev., 2003, 32, pp. 268–275
8. Lancaster. K.M. et al.; Science, 2011: 334 (6058), pp. 974-977
9. Goodsell, D.; PDB, February 2002, Molecule of the Month   

           All protein figures generated using PyMol, from PDB structure 1N2C.

Literature Summaries

Unraveling the molecular mechanisms of nitrogenase conformational protection against oxygen in diazotrophic bacteria

Nitrogenase serves as the primary protein in the nitrogen fixation process, converting molecular nitrogen to ammonia. In most cases, oxygen irreversibly inhibits the function of nitrogenase; however, as the protein functions in aerobic environments, there is more to the story. This paper explores the sequence homology of the nitrogenase protein to other oxygen-sensitive diazotropic bacteria, revealing a method of conformational protection on the main domain. In addition, surface analysis revealed an electrostatic surface dimerization domain, where two FeS protein subunits can associate using a ‘beta-grasp’ mechanism to prevent oxygen damage to the reactive FeS metallo-cluster. In the process, the protein-coding genes were isolated in A. vinelandii.

BMC Genomics 2010, 11(Suppl 5):S7

Nitrogenase MoFe-Protein at 1.16 Å Resolution: A Central Ligand in theFeMo-Cofactor

Nitrogenase incorporates an Iron-Sulfur cluster as a cofactor for catalytic activity. The protein performs intra-protein electron transfer from the homodimeric Fe metalloprotein, via the hydrolysis of adenosine 5’-triphosphate, to the MoFe protein (an α₂β₂ tetramer). It is thought that dinitrogen is reduced in the active site of the FeMo cofactor, which is examined closely in this study. The authors observed six central iron atoms which lie on the surface of a sphere with a radius of 2.0 Å from the cofactor center, coordinated to three inorganic sulfur atoms. These sulfur atoms of the FeMo-cofactor are themselves equidistant from the center on a second sphere with a radius of 3.3 Å. The central pocket of this cluster contains an unidentified atom with a fully occupied valence, closer inspection identifying it as nitrogen. The geometry enforced by the structure of the cofactor provides optimal arrangements for dinitrogen reduction.


Update: In a recent publication, the central ligand of the FeMoco cofactor was identified as a µ₆Carbide...
yes, that is 6 bonds to Carbon! This was identified by Lancaster et. al. using Fe-KEdge spectroscopy and
is reported here.

Science , New Series, Vol. 297, No. 5587 (Sep. 6, 2002), pp. 1696-1700

Climbing Nitrogenase: Toward a Mechanismof Enzymatic Nitrogen Fixation

N2 + 8e- + 16MgATP + 8H+ ⇒ 2NH3 + H2 + 16MgADP + 16Pi                         (1)

Figure 4. “Alternating” (A) and “distal” (D) N₂ reduction pathways. M represents FeMo-co without specifying metal ion(s) involved. Likewise, the representations of binding are meant to emphasize the distinction between pathways and do not imply specific binding modes. Small straight arrows represent addition of H⁺/e⁻ to substrate.

This paper addresses the reaction mechanism and pathway promoted by nitrogenase. Evidently, the 8 electron oxidation is both thermally and kinetically disfavored, leading to the analogy of scaling a peak. The two viable paths to the ‘top’ of the energy barrier are
(a) “distal”: by sequentially adding electrons to each individual nitrogen atom, or
(b) “alternating”: by interchanging between the two atoms of N₂. 

In addition, the peak analogy persists when considering the difficulty in trapping reaction intermediates to identify substrate-bound species, especially those from the late stages of the reduction pathway. The identification of the reaction pathway is compounded by the mystery of the number, and order, of electrons and protons being delivered to the Mo-Fe cluster during the reaction.

Chart 1. Substrate-Derived Species Bound to FeMo-co That Might Form in Late Stages of N₂ Reduction

Nitrogenase Images

All figures from PDB: 1N2C, created using PyMol.

MoFe Protein active site in central location of protein, flanked by two Fe-protein chain subunits on either side.

Add caption

Active Site of Fe protein subunit. 

FeS cofactor cluster, used to transport electrons to the MoFe protein subunit. 

Active Site of Fe-subunit.