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Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08 Å resolution: comparison with the Azotobacter vinelandii MoFe protein.

Zhang LM, Morrison CN, Kaiser JT, Rees DC - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction.The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor.This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

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Characterization of the interstitial ligand in the Cp1 FeMo cofactor. (a) The refined crystallographic structure of the FeMo cofactor with the superimposed 2Fo − Fc electron-density map highlighted in light blue and contoured at 3σ. The interstitial ligand is modeled as carbon and labeled CX. C atoms are shown in light gray, N atoms in blue, O atoms in red, S atoms in yellow, Fe atoms in orange and Mo atoms in cyan. Homocitrate is labeled HCA. (b) The averaged electron density ρ(r) of the two crystallographically independent interstitial ligands CX (green) calculated within spheres of the indicated radii and compared with those calculated for proteinaceous C (gray), N (blue) and O (red) atoms with an isotropic B factor no greater than 30 Å2. (c) The variation in electron density (ρ0) at the atomic center as a function of the isotropic B factor for proteinaceous carbon (gray), nitrogen (blue) and oxygen (red). Atoms with isotropic B factors of <30 Å2 were included in the calculation. The data points representing two crystallographically independent interstitial ligands CX are shown in green.
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fig2: Characterization of the interstitial ligand in the Cp1 FeMo cofactor. (a) The refined crystallographic structure of the FeMo cofactor with the superimposed 2Fo − Fc electron-density map highlighted in light blue and contoured at 3σ. The interstitial ligand is modeled as carbon and labeled CX. C atoms are shown in light gray, N atoms in blue, O atoms in red, S atoms in yellow, Fe atoms in orange and Mo atoms in cyan. Homocitrate is labeled HCA. (b) The averaged electron density ρ(r) of the two crystallographically independent interstitial ligands CX (green) calculated within spheres of the indicated radii and compared with those calculated for proteinaceous C (gray), N (blue) and O (red) atoms with an isotropic B factor no greater than 30 Å2. (c) The variation in electron density (ρ0) at the atomic center as a function of the isotropic B factor for proteinaceous carbon (gray), nitrogen (blue) and oxygen (red). Atoms with isotropic B factors of <30 Å2 were included in the calculation. The data points representing two crystallographically independent interstitial ligands CX are shown in green.

Mentions: A significant advantage of atomic resolution crystal structures is to minimize the influence of series-termination effects in Fourier maps, which are of particular concern for the interstitial ligand of the FeMo cofactor, as it is surrounded by six equidistant irons (Spatzal et al., 2011 ▶; Einsle et al., 2002 ▶). In the 2Fo − Fc map of the 1.08 Å resolution Cp1 structure, the electron density at the center of the FeMo cofactor clearly indicates the presence of an interstitial ligand in the cofactor of Cp1 (Fig. 2 ▶a). Using the electron-density analyses developed previously (Spatzal et al., 2011 ▶), the interstitial ligand has similar properties in both Cp1 and Av1, consistent with the assignment of this atom as carbon. Comparison of the averaged electron density ρ(r) calculated within spheres of different radii around a given atom type shows that the interstitial ligand in the FeMo cofactor most closely resembles proteinaceous carbon, but not nitrogen or oxygen (Fig. 2 ▶b). The deviation of ρ(r) for the interstitial ligand from that of proteinaceous carbon at larger radius (>0.8 Å) may reflect the truncation error caused by the surrounding heavy atoms, such as Fe atoms, which are about 2 Å away from the interstitial ligand. In addition, the correlation between the electron density at the center of a given type of atom and the isotropic B factor of the interstitial ligand also falls in the range of proteinaceous carbon, but not nitrogen or oxygen (Fig. 2 ▶c).


Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08 Å resolution: comparison with the Azotobacter vinelandii MoFe protein.

Zhang LM, Morrison CN, Kaiser JT, Rees DC - Acta Crystallogr. D Biol. Crystallogr. (2015)

Characterization of the interstitial ligand in the Cp1 FeMo cofactor. (a) The refined crystallographic structure of the FeMo cofactor with the superimposed 2Fo − Fc electron-density map highlighted in light blue and contoured at 3σ. The interstitial ligand is modeled as carbon and labeled CX. C atoms are shown in light gray, N atoms in blue, O atoms in red, S atoms in yellow, Fe atoms in orange and Mo atoms in cyan. Homocitrate is labeled HCA. (b) The averaged electron density ρ(r) of the two crystallographically independent interstitial ligands CX (green) calculated within spheres of the indicated radii and compared with those calculated for proteinaceous C (gray), N (blue) and O (red) atoms with an isotropic B factor no greater than 30 Å2. (c) The variation in electron density (ρ0) at the atomic center as a function of the isotropic B factor for proteinaceous carbon (gray), nitrogen (blue) and oxygen (red). Atoms with isotropic B factors of <30 Å2 were included in the calculation. The data points representing two crystallographically independent interstitial ligands CX are shown in green.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4321486&req=5

fig2: Characterization of the interstitial ligand in the Cp1 FeMo cofactor. (a) The refined crystallographic structure of the FeMo cofactor with the superimposed 2Fo − Fc electron-density map highlighted in light blue and contoured at 3σ. The interstitial ligand is modeled as carbon and labeled CX. C atoms are shown in light gray, N atoms in blue, O atoms in red, S atoms in yellow, Fe atoms in orange and Mo atoms in cyan. Homocitrate is labeled HCA. (b) The averaged electron density ρ(r) of the two crystallographically independent interstitial ligands CX (green) calculated within spheres of the indicated radii and compared with those calculated for proteinaceous C (gray), N (blue) and O (red) atoms with an isotropic B factor no greater than 30 Å2. (c) The variation in electron density (ρ0) at the atomic center as a function of the isotropic B factor for proteinaceous carbon (gray), nitrogen (blue) and oxygen (red). Atoms with isotropic B factors of <30 Å2 were included in the calculation. The data points representing two crystallographically independent interstitial ligands CX are shown in green.
Mentions: A significant advantage of atomic resolution crystal structures is to minimize the influence of series-termination effects in Fourier maps, which are of particular concern for the interstitial ligand of the FeMo cofactor, as it is surrounded by six equidistant irons (Spatzal et al., 2011 ▶; Einsle et al., 2002 ▶). In the 2Fo − Fc map of the 1.08 Å resolution Cp1 structure, the electron density at the center of the FeMo cofactor clearly indicates the presence of an interstitial ligand in the cofactor of Cp1 (Fig. 2 ▶a). Using the electron-density analyses developed previously (Spatzal et al., 2011 ▶), the interstitial ligand has similar properties in both Cp1 and Av1, consistent with the assignment of this atom as carbon. Comparison of the averaged electron density ρ(r) calculated within spheres of different radii around a given atom type shows that the interstitial ligand in the FeMo cofactor most closely resembles proteinaceous carbon, but not nitrogen or oxygen (Fig. 2 ▶b). The deviation of ρ(r) for the interstitial ligand from that of proteinaceous carbon at larger radius (>0.8 Å) may reflect the truncation error caused by the surrounding heavy atoms, such as Fe atoms, which are about 2 Å away from the interstitial ligand. In addition, the correlation between the electron density at the center of a given type of atom and the isotropic B factor of the interstitial ligand also falls in the range of proteinaceous carbon, but not nitrogen or oxygen (Fig. 2 ▶c).

Bottom Line: The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction.The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor.This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

Show MeSH
Related in: MedlinePlus