Nitrogenase Complex

Abstract

Nitrogenases are a family of metalloenzymes that catalyse a key step in the global nitrogen cycle: the adenosine triphosphate (ATP)‐dependent reduction of dinitrogen (N2) to ammonia (NH3). The molybdenum (Mo)‐nitrogenase is the best characterised member of this enzyme family. It is a two‐component system comprising an iron (Fe) protein and a molybdenum‐iron (MoFe) protein, each containing FeS cluster(s) which are responsible for the electron flow during the process of substrate reduction. This article provides an overview of the current knowledge on the structure, assembly and catalysis of Mo‐nitrogenase, as well as a brief discussion of alternative substrates of nitrogenase and other members in this enzyme family.

Key Concepts:

  • The nitrogenase complex consists of two Fe protein dimers and one MoFe protein tetramer; each component harbours metallocluster(s) that mediate electron flow to the active centre within the complex.

  • The active centre of Mo‐nitrogenase, designated the FeMoco or the M‐cluster, is a [MoFe7S9C‐homocitrate] cluster.

  • Electrons are transferred from the [Fe4S4] cluster in the Fe protein to the P‐cluster and then the M‐cluster in the MoFe protein, where substrate reduction occurs.

  • Nitrogenase catalyses the ATP‐dependent reduction of dinitrogen (N2) to ammonia (NH3) under ambient temperature and pressure.

  • Nitrogenase has been found to catalyse the reduction of alternative substrates, such as H+, N3, CN, C2H2 and CO.

Keywords: nitrogenase; nitrogen fixation; MoFe protein; Fe protein; P‐cluster; FeMoco; M‐cluster; L‐cluster; K‐cluster

Figure 1.

Crystal structure of the Mo‐nitrogenase complex. MoFe protein and Fe protein are locked in complex by the nonhydrolysable ATP analogue, MgADP·AlF4−. The structure is rendered semitransparent to allow visualisation of electron flow from the [Fe4S4] cluster in Fe protein, through the P‐cluster, to the M‐cluster in MoFe protein, which is the site of substrate reduction. The subunits of Fe protein are shown in blue; and the α‐ and β‐subunits of MoFe protein are shown in brown and green, respectively. Clusters are shown in ball‐and‐stick presentations. Atoms are coloured as follows: Fe, orange; S, yellow; Mo, cyan; O, red; C, grey; N, dark blue; Mg, green; Al, beige and F, light blue. PYMOL was used to create this figure using PDB entry 1M34.

Figure 2.

Assembly of P‐cluster. (a) The two P‐clusters in MoFe protein are sequentially assembled in situ. NifU and NifS supply two [Fe4S4]‐like cluster pairs to the MoFe protein, one pair at each α/β‐subunit interface. The formation of the first P‐cluster requires Fe protein, MgATP and reductant, whereas the formation of the second P‐cluster also requires the action of NifZ. The half‐assembled (II) and fully‐assembled (III) conformations can be achieved by incubating the ΔnifH MoFe protein (I) with the Fe protein, ATP and reductant with increasing time; alternatively, they can be captured through genetic manipulations in the ΔnifBZ and ΔnifB MoFe proteins, respectively. The formation of P‐cluster also induces a conformational change of the α‐subunit, which ‘opens’ up the M‐cluster site. (b) Conversion of a P‐cluster precursor (a pair of [Fe4S4]‐like clusters) to a mature P‐cluster (an [Fe8S7] cluster) via reductive coupling. Clusters are shown in ball‐and‐stick presentations. Atoms are coloured as follows: Fe, orange; S, yellow; C, grey and O, red. PYMOL was used to create portions of this figure.

Figure 3.

Assembly of M‐cluster. (a) Combined actions of NifS and NifU lead to the sequential formation of [Fe2S2] and [Fe4S4] clusters. Subsequently, the [Fe4S4] clusters are delivered to NifB, where a pair of [Fe4S4] clusters (K‐cluster) is converted to an [Fe8S9C] cluster (L‐cluster). The L‐cluster is then transferred to NifEN, a structural homologue to MoFe protein (see Figure a), and matured into an [MoFe7S9C‐homocitrate] (M‐cluster) in an Fe protein/ATP‐dependent process. Finally, the M‐cluster is transferred to apo‐MoFe protein via direct protein–protein interactions and inserted into the binding site via a positively charged insertion funnel (see Figure b). (b) The assembly of an M‐cluster from small FeS building blocks. First, elemental Fe and S (Cys) are mobilised by NifS and NifU for the sequential formation of [Fe2S2] and [Fe4S4] clusters. Then, the two [Fe4S4] units of K‐cluster are coupled into an L‐cluster ([Fe8S9C]) on NifB concomitant with the insertion of a S atom (of unknown origin) and a C atom (from the methyl group of SAM) via radical chemistry. Finally, the L‐cluster ([Fe8S9C]) is matured on NifEN into an M‐cluster ([MoFe7S9C‐homocitrate]) via ATP‐dependent insertion of Mo and homocitrate by Fe protein. Clusters are shown in ball‐and‐stick presentations. Atoms are coloured as follows: Fe, orange; S, yellow; Mo, cyan; O, red and C, grey. PYMOL was used to generate portions of this figure.

Figure 4.

Structures of NifEN, MoFe protein and apo‐MoFe protein. (a) Ribbon presentations of the αβ‐dimers of NifEN (left) and MoFe protein (right), showing the location of the FeS cluster species within each protein. The α‐subunits are coloured blue and presented in the foreground, whereas the β‐subunits are rendered transparent in the background. (b) Ribbon presentation of the αβ‐dimer of apo‐MoFe protein (left) and its corresponding electrostatic surface potential presentation (right), revealing the presence of a positively charged M‐cluster insertion funnel (coloured blue). Key residues in the insertion funnel (viewed from the top) are shown in the dashed circle (b, right). All clusters are shown in space‐filling models, with atoms coloured as follows: Fe, orange; S, yellow; Mo, cyan; O, red and C, grey. PYMOL was used to generate this figure using coordinates of PDB entries 1L5H, 3PDI and 1M1N.

Figure 5.

Catalysis by Mo‐nitrogenase. (a) The Fe protein cycle. This cycle consists of the following steps: (i) the formation of a complex between the ATP‐bound Fe protein and the MoFe protein; (ii) the transfer of electrons from the Fe protein to the MoFe protein concomitant with the hydrolysis of ATP; (iii) the dissociation of the complex following the release of phosphate (Pi) and (iv) the rereduction of the Fe protein and the replacement of ADP with ATP. FePred, reduced Fe protein; FePOX, oxidised Fe protein; MoFePred, reduced MoFe protein. (b) The MoFe protein cycle. This cycle consists of a number of steps, where e/H+ pairs are added one at a time on the MoFe protein, converting it from the resting state (M0) to the turnover state (Mn). The binding of N2 likely occurs at the M3 stage, where three e/H+ pairs have been accumulated concomitant with the release of H2. The distal N in the N2 molecule is reduced and released as NH3 at the M4 stage; whereas the proximal N is reduced and released as NH3 at the M7 stage. n, number of e/H+ pairs added on the MoFe protein.

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Further Reading

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Wiig JA, Lee CC, Fay AW, Hu Y and Ribbe MW (2011) Purification of nitrogenase proteins. Methods in Molecular Biology 766: 93–103.

Yang ZY, Danyal K and Seefeldt LC (2011) Mechanism of Mo‐dependent nitrogenase. Methods in Molecular Biology 766: 9–29.

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Wiig, Jared A, Rebelein, Johannes G, and Hu, Yilin(May 2014) Nitrogenase Complex. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001386.pub2]