Molecular insights into nitrogenase FeMoco insertion--the role of His 274 and His 451 of MoFe protein alpha subunit

Abstract

The final step of FeMo cofactor (FeMoco) assembly involves the insertion of FeMoco into its binding site in the molybdenum-iron (MoFe) protein of nitrogenase. Here we examine the role of His alpha274 and His alpha451 of Azotobacter vinelandii MoFe protein in this process. Our results from combined metal, activity, EPR, stability and insertion analyses show that mutations of His alpha274 and/or His alpha451, two of the histidines that belong to a so-called His triad, to small uncharged Ala specifically reduce the accumulation of FeMoco in MoFe protein. This observation indicates that the enrichment of histidines at the His triad is important for FeMoco insertion and that the His triad potentially serves as an intermediate docking point for FeMoco through transitory ligand coordination and/or electrostatic interaction.

Fig. 1

Overlaid protein environment of Av1^wild-type and Av1^ΔnifB in the vicinity of the FeMoco binding site. Parts of the C_α backbones of Av1^wild-type and Av1^ΔnifB are shown and colored as follows: Av1^wild-type in green and Av1^ΔnifB in light grey. FeMoco is shown in ball-and-stick presentation and colored as follows: oxygen in red, carbon in dark grey, molybdenum in orange, sulfur in yellow, iron in magenta. Note that, from this angle, only a small portion of the molybdenum atom is visible, while the central atom X (of unknown origin) is invisible. The side chains of His α274, His α451, His α442 and Cysα275 are shown in line presentation and colored as follows: His α274, His α451, His α442 of Av1^wild-type in green and blue, His α274, His α451, His α442 of Av1^ΔnifB in light grey and blue, Cys α275 of Av1^wild-type in green and yellow, and Cys α275 of Av1^ΔnifB in light grey and yellow. His α442 and Cys α275 are the two protein ligands that are covalently attached to FeMoco in Av1^wild-type. In Av1^ΔnifB, the position of Cys α275 remains largely unchanged, whereas His α442 is re-positioned by a distance of approximately 5 Å and joins His α451 and His α274 in the formation of a striking His triad, which presumably serves to guide the FeMoco to its final location. The distances between the His triad residues, measured by the distances between their respective C2 atoms of the imidazole rings (the carbon atom between the two ring nitrogen atoms) are: (i) 3.62 Å between His α451 and His α274; (ii) 3.60 Å between His α274 and His α442; and (iii) 3.66 Å between His α442 and His α451. Additionally, His α442 can potentially form a hydrogen bond with the nearby Asp α234.

Fig. 2

Coomassie stained SDS-PAGE (A) and Western blots using Av2-specific (B) and Av1-specific (C) antibodies against cell-free crude extracts of A. vinelandii strains. (A) Lane 1, protein standard, 15 μg; lane 2, AvYM13A^wild-type crude extract, 20 μg; lane 3, AvYM14A^αH274A crude extract, 20 μg; lane 4, AvYM22A^αH451A crude extract, 20 μg; lane 5, AvYM23A^{αH274A/αH451A} crude extract, 20 μg; lane 6, purified Av1^wild-type, 3.5 μg; lane 7, purified Av2^wild-type, 3.5 μg. (B) Lane 1, AvYM13A^wild-type crude extract, 20 μg; lane 2, AvYM14A^αH274A crude extract, 20 μg; lane 3, AvYM22A^αH451A crude extract, 20 μg; lane 4, AvYM23A^{αH274A/αH451A} crude extract, 20 μg; lane 5, purified Av2^wild-type, 3.5 μg. (C) Lane 1, AvYM13A^wild-type crude extract, 20 μg; lane 2, AvYM14A^αH274A crude extract, 20 μg; lane 3, AvYM22A^αH451A crude extract, 20 μg; lane 4, AvYM23A^{αH274A/αH451A} crude extract, 20 μg; lane 5, purified Av1^wild-type, 3.5 μg. Note that Western blot analyses were performed under non-saturated conditions.

Fig. 3

Coomassie stained SDS-PAGE of purified Av2 (A) and Av1 (B) proteins. (A) 10–20% gradient SDS-PAGE of Av2 proteins: lane 1, protein standard, 15 μg; lane 2, Av2 ^wild-type, 4 μg; lane 3, Av2^αH274A, 4 μg; lane 4, Av2^αH451A, 4 μg; lane 5, Av2^{αH274A/αH451A}, 4 μg. The molecular masses of all purified Av2 proteins in this study appeared to be identical based on their elution profiles on gel filtration Sephacryl S-200 HR columns (Amersham Biosciences). (B) 4–15% gradient SDS-PAGE of Av1 proteins: lane 1, protein standard, 15 μg; lane 2, Av1 ^wild-type, 5 μg; lane 3, Av1^αH274A, 5 μg; lane 4, Av1^αH451A, 5 μg; lane 5, Av1^{αH274A/αH451A}, 5 μg. The molecular masses of all purified Av1 proteins in this study appeared to be identical based on their elution profiles on gel filtration Sephacryl S-200 HR columns (Amersham Biosciences).

Fig. 4

Perpendicular mode EPR spectra of dithionite-reduced Av2^wild-type (1), Av2^αH274A (2), Av2^αH451A (3) and Av2^{αH274A/αH451A} (4). EPR samples (20 mg/ml) were prepared and measured as described in “Materials and methods”. All Av2 proteins show the characteristic S = 1/2 signal of the [4Fe-4S]¹⁺ cluster at nearly the same intensity.

Fig. 5

(A) Parallel mode EPR spectra of IDS-oxidized Av1^wild-type (1), Av1^αH274A (2), Av1^αH451A (3) and Av1^{αH274A/αH451A} (4). EPR samples (20 mg/ml) were prepared and measured as described in “Materials and methods”. The P-cluster specific (P²⁺ state), g = 11.8, parallel mode EPR signals of Av1^αH274A, Av1^αH451A and Av1^{αH274A/αH451A} integrate to 97%, 97% and 95% of that of Av1^wild-type, respectively. (B) Perpendicular mode EPR spectra of dithionite-reduced Av1^wild-type (1), Av1^αH274A (2), Av1^αH451A (3) and Av1^{αH274A/αH451A} (4). EPR samples (20 mg/ml) were prepared and measured as described in “Materials and methods”. The FeMoco-specific, S = 3/2 EPR signals of Av1^αH274A, Av1^αH451A and Av1^{αH274A/αH451A} integrate to 44%, 58% and 29% of that of Av1^wild-type, respectively. The S = 1/2 EPR signals in the g ≈ 2 region of the spectra of Av1^αH274A, Av1^αH451A and Av1^{αH274A/αH451A} integrate to approximately 0.04, 0.05 and 0.10 spin per protein, respectively. The g values are indicated.

Fig. 6

Temperature- (A) and power- (B) dependency of the perpendicular mode EPR signals exhibited by dithionite-reduced Av1^wild-type (1), Av1^αH274A (2), Av1^αH451A (3) and Av1^{αH274A/αH451A} (4). All spectra were measured at a protein concentration of 20 mg/ml between 6 and 20 K at 50 mW (A) or between 5 and 50 mW at 10 K (B) as described in “Materials and methods”. The g values are indicated.

Fig. 7

Protection of chelation of iron from the [4Fe-4S]¹⁺ cluster of Av2^wild-type by complex formation of Av2^wild-type with Av1^wild-type (2), Av1^αH274A (3), Av1^αH451A (4) and Av1^{αH274A/αH451A} (5). Formation of the complex between the iron chelator bathophenanthroline disulfonate and the iron from the [4Fe-4S]¹⁺ cluster of the Av2^wild-type was measured at 535 nm in the presence of MgATP alone (1), MgATP plus Av1 proteins (2–5), or MgADP alone (6). Final concentrations of 0.4 mM MgCl₂ and 0.2 mM of either ADP or ATP were used. The concentrations of Av1 and Av2 proteins were 0.35 and 0.2 mg/ml, respectively. Curves obtained in the presence of MgATP and Av1 proteins were fitted to single exponential equations over a period of 20 s, giving the following observed rate constants: 0.0014 s⁻¹ (1), 0.0008 s⁻¹ (2), 0.0009 s⁻¹ (3), 0.0008 s⁻¹ (4), 0.0009 s⁻¹ (5) and 0 s⁻¹ (6). These studies show that each His triad Av1 variant protein forms a complex with Av2^wild-type that is similarly stable compared to that formed between Av1^wild-type and Av2^wild-type.

Fig. 8

Time-dependent FeMoco insertion into Av1^ΔnifB (-●-), Av1^{ΔnifB/αH274A} ( ), Av1^{ΔnifB/αH451A} ( ) and Av1^{ΔnifB/αH274A/αH451A} (-∇-). FeMoco insertion was carried out as described in “Materials and methods”. The inset shows the first minute of the insertion, giving observed rates at 63% (Av1^{ΔnifB/αH274A}), 54% (Av1^{ΔnifB/αH451A}) and 36% (Av1^{ΔnifB/αH274A/αH451A}), respectively, of that of Av1^ΔnifB (set at 100%).