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Last modified: 2 February 1999

Mo­nitrogenase component I (MoFe protein)
Metal cluster Formal oxidation/spin states
P
Pn cluster image
[Fe8S7](SgammaCys)6		 [Fe8S7]N (S=0);

[Fe8S7]SEMI­OX (S=1/2; S=5/2)
Pox cluster image
[Fe8S7](SgammaCys)6NCysOgammaSer		 [Fe8S7]OX1 (S=3; S=4);

[Fe8S7]OX2 (S=1/2; S=7/2)

M
M-cluster image
([Fe7MoS9]·homocitrate)SgammaCysNdeltaHis		 [Fe7MoS9]R (S=integer);

[Fe7MoS9]N (S=3/2);

[Fe7MoS9]OX (S=0)

Biological nitrogen fixation, i.e. reduction of molecular nitrogen to ammonia (1), is catalysed by the nitrogenase enzyme system (EC 1.18.6.1). Molybdenum nitrogenase (Mo­nitrogenase), which is found in all nitrogen fixing organisms, consists of two components: component I [nitrogenase molybdenum-iron (MoFe) protein, or dinitrogenase], and component II [nitrogenase iron (Fe) protein, or dinitrogenase reductase] [1; see list of reviews on structure and function of Mo­nitrogenase].

The MoFe protein is an alpha2ß2 tetramer; the alpha­ and ß­subunits are products of the nifD and nifK genes respectively. The Fe protein is a homodimer, the monomer being coded for by the nifH gene. Nitrogenase binds and hydrolyses 2MgATP, yielding 2MgADP and 2Pi for each electron that is transferred from the Fe protein to the MoFe protein [2]. The MoFe protein contains two types of metal centres, called the M­cluster and the P­cluster. The M­cluster, also termed the iron-molybdenum cofactor (FeMoco), is thought to provide the substrate­binding site. Biosynthesis of FeMoco in vivo requires at least six nif gene products [3]. The M­cluster can adopt at least three oxidation states, usually referred to as native (semi­reduced, MN), oxidised (MOX) and reduced (MR). The P­clusters can reversibly achieve four oxidation states, from native (reduced) MN to MOX2. The net charge on the cluster at any oxidation level is unknown [1]. The function of the P­cluster may be the electron transfer between the [Fe4S4] centre of the Fe protein and the FeMoco. Thus, the general sequence of electron transfer steps in nitrogenase system appears to be It also has been proposed that loss of P­clusters alters the catalytic properties of FeMoco or allows catalytically inactive FeMoco or FeMoco­like species to be incorporated at the M­cluster site of the MoFe protein [4].

The 3­D structures of Azotobacter vinelandii and Clostridium pasteurianum nitrogenase MoFe proteins have been determined [5, 6]. The alpha2ß2 tetramer consists of two alphaß dimers that are related by the molecular 2­fold rotation axis (see Figure 2MIN a). alpha­ and ß­subunits in an alphaß dimer are also roughly related by a 2­fold rotation (Figure 2MIN b). The alpha­ and ß­subunits exhibit similar folds consisting of three alpha/ß domains (Figure 2MIN c, 2MIN d). In each subunit, there is a cleft between the three domains. The FeMoco resides at the bottom of this cleft in the alpha­subunit. FeMoco consists of two cuboidal fragments, [Fe4S3] and [Fe3MoS3], which are bridged by three sulphur atoms, and homocitrate [(R)­2­hydroxybutane­1,2,4­tricarboxylic acid]. The Mo is octahedrally coordinated, whereas Fe atoms at the interface of the two cuboidal fragments have open coordination sites and supposedly bind N2. FeMoco is attached to the alpha­subunit by two residues (Cys­275 and His­442 in the A. vinelandii MoFe protein) (Figure 2MIN e, left).

The P­cluster is located on the pseudo 2­fold axis that relates the alpha­ and ß­subunits. Previously, an [Fe8S8] model for the P­cluster was proposed, where two nearly symmetrical [Fe4S4] cubes were held together by two bridging Cys residues and a single disulphide bond between two sulphur atoms of the two cubes [5]:

However, using crystals of the MoFe protein characterised with respect to oxidation state, it was found that the P­cluster in both oxidation states exists as an [Fe8S7] cluster [7] (see Figure 2MIN e, g and h). The P­cluster may be described as containing bridged [Fe4S4] and [Fe4S3] clusters. Two bridging Cys residues originate from each of the alpha­ and ß­subunits (Cysalpha­88 and Cysß­95 in the A. vinelandii MoFe protein). (Interestingly, the site­directed mutagenesis studies of Klebsiella pneumoniae nitrogenase MoFe protein show that these two Cys residues are not essential for maintaining the integrity of the P­clusters [4]). Apart from the two bridging Cys residues, each cube of the P­cluster is attached either to the alpha­ or to the ß­subunit through two Cys residues (Cysalpha­62, Cysalpha­154, Cysß­70, Cysß­153 in the A. vinelandii MoFe protein). In the oxidised state (POX), two additional protein ligands to the P­cluster are present, viz. a backbone amide nitrogen of Cysalpha­88 and the Ogamma of Serß­188: In the reduced state (PN), these two non­cysteinyl ligands are replaced by interactions with the central sulphur atom which now adopts a distorted octahedral coordination geometry (cf. distorted tetrahedral geometry in POX): Both M­ and P­clusters are buried about 10 Å below the protein surface [5]. In the crystal structure of the MoFe protein-Fe protein complex, the P­cluster is located equidistant (~14 Å) between the [Fe4S4] cluster of the Fe protein and FeMoco [8]. This arrangement strongly suggests that electrons are transferred from the Fe protein to the FeMoco via the P­cluster (2).

Nitrogenase in enzyme databases

ENZYME LIGAND BRENDA Official name
1.18.6.1
1.18.6.1
1.18.6.1
 Nitrogenase

MoFe protein in motif databases

PRINTS ID PRINTS AC PROSITE/BLOCKS ID PROSITE AC BLOCKS AC
-
-
 NITROGENASE_1_1
NITROGENASE_1_2		 PS00699
PS00090		 BL00699

MoFe protein in alignment databases

Protein Superfamily Protein Homology Domain Pfam LPFC 3­D
alignment
00215; dinitrogenase alpha chain 00311; nitrogenase MoFe / VFe protein alpha chain
PF00148; oxidored_nitro
-
00216; dinitrogenase ß chain
-
-

MoFe protein in 3­D databases

Nitrogenase component I (MoFe protein) tetramer binds two M­clusters (FeMoco) and two P­clusters (see Figure 2MIN).

PDB scop BSMRELI
Base		 Header MMS Abstract ¹
1mio 1mio 1mio 1mio Nitrogenase MoFe protein; Clostridium pasteurianum MMS94189
1n2c 1n2c 1n2c 1n2c Nitrogenase complex (2:1 complex of homodimeric Fe protein and alpha2ß2 heterotetrameric MoFe protein) (complex with ADP, AlF4¯, Ca2+, Mg2+ and 3­hydroxy­3­carboxy­adipic acid); Azotobacter vinelandii
-
2min 2min 2min 2min Nitrogenase MoFe protein (POX/MOX) (complex with Ca2+ and 3­hydroxy­3­carboxy­adipic acid); Azotobacter vinelandii MMS93147
3min 3min 3min 3min Nitrogenase MoFe protein (PN/MN) (complex with Ca2+ and 3­hydroxy­3­carboxy­adipic acid); Azotobacter vinelandii
-

¹ Macromolecular Structures abstract. Full text is available to BioMedNet Members

References

  1. Burgess, B.K. and Lowe, D.L. (1996) Mechanism of molybdenum nitrogenase. Chem. Rev. 96, 2983-3012.
  2. Seefeldt, L.C., Morgan, T.V., Dean, D.R. and Mortenson, L.E. (1992) Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction. J. Biol. Chem. 267, 6680-6688.
  3. Allen, R.M., Chatterjee, R., Madden, M.S., Ludden, P.W. and Shah, V.K. (1994) Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Crit. Rev. Biotechnol. 214, 225-249.
  4. Yousafzai, F.K., Buck, M. and Smith, B.E. (1996) Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P­clusters are replaced by the non­coordinating amino acid alanine. Biochem. J. 318, 111-118.
  5. Kim, J. and Rees, D.C. (1992) Crystallographic structure and functional implications of the nitrogenase molybdenum-iron protein from Azotobacter vinelandii. Nature 360, 553-560.
  6. Kim, J., Woo, D. and Rees, D.C. (1993) X­ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0­Å resolution. Biochemistry 32, 7104-7115.
  7. Peters, J.W., Stowell, M.H., Soltis, S.M., Finnegan, M.G., Johnson, M.K. and Rees, D.C. (1997) Redox­dependent structural changes in the nitrogenase P­cluster. Biochemistry 36, 1181-1187.
  8. Schindelin, H., Kisker, C., Schlessman, J.L., Howard, J.B. and Rees, D.C. (1997) Structure of ADP·AlF4¯­stabilized nitrogenase complex and its implications for signal transduction. Nature 387, 370-376.
Bibliography on structural studies of Mo­nitrogenase component I
Reviews on Mo­nitrogenase