Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism

Abstract

NifEN is a key player in the biosynthesis of nitrogenase MoFe protein. It not only shares a considerable degree of sequence homology with the MoFe protein, but also contains clusters that are homologous to those found in the MoFe protein. Here we present an investigation of the catalytic activities of NifEN. Our data show that NifEN is catalytically competent in acetylene (C(2)H(2)) and azide (N(3)(-)) reduction, yet unable to reduce dinitrogen (N(2)) or evolve hydrogen (H(2)). Upon turnover, C(2)H(2) gives rise to an additional S = 1/2 signal, whereas N(3)(-) perturbs the signal originating from the NifEN-associated FeMoco homolog. Combined biochemical and spectroscopic studies reveal that N(3)(-) can act as either an inhibitor or an activator for the binding and/or reduction of C(2)H(2), while carbon monoxide (CO) is a potent inhibitor for the binding and/or reduction of both N(3)(-) and C(2)H(2). Taken together, our results suggest that NifEN is a catalytic homolog of MoFe protein; however, it is only a "skeleton" version of the MoFe protein, as its associated clusters are simpler in structure and less versatile in function, which, in turn, may account for its narrower range of substrates and lower activities of substrate reduction. The resemblance of NifEN to MoFe protein in catalysis points to a plausible, sequential appearance of the two proteins in nitrogenase evolution. More importantly, the discrepancy between the two systems may provide useful insights into nitrogenase mechanism and allow reconstruction of a fully functional nitrogenase from the "skeleton" enzyme, NifEN.

Fig. 1.

Electron transfer during nitrogenase catalysis. (A) Crystal structure of MgADP·AlF₄⁻-stabilized complex between Fe protein and one αβ-dimer of MoFe protein. (B) Electron transfer pathway between Fe protein and MoFe protein. (C) Hypothetical electron transfer pathway between Fe protein and NifEN. It has been proposed that, during nitrogenase catalysis, electrons flow from the [Fe₄S₄] cluster of the Fe protein to the P-cluster ([Fe₈S₇]) and then the FeMoco ([MoFe₇S₉X-homocitrate], where X = C, N, or O) of MoFe protein. Likewise, electrons could flow from the [Fe₄S₄] cluster of the Fe protein to the [Fe₄S₄] cluster and then the FeMoco homolog of NifEN. Figures are generated in PYMOL using 1N2C and 1M1N PDB coordinates (3, 21). The two subunits of Fe protein are colored yellow and orange, and the α- and β-subunits of MoFe protein are colored blue and violate. Atoms of clusters are colored as follows: Fe, purple; S, green; Mo, burgundy; C, dark gray; and O, red. Note that homocitrate is missing from the NifEN-associated FeMoco homolog, and Mo is either absent or replaced by Fe (colored light gray) in the FeMoco homolog structure.

Fig. 2.

Substrate-reducing activities of NifEN. (A and B) Dependence of C₂H₂ (A) and N₃⁻ (B) reducing activities on dithionite concentration. (C and D) Dependence of C₂H₂ (C) and N₃⁻ (D) reducing activities on Fe protein/NifEN ratio. (E and F) Dependence of C₂H₂ (E) and N₃⁻ (F) reducing activities on ATP hydrolysis. AMPPNP, ATPγS, non-hydrolysable ATP analogs. A157S Fe protein, a Fe protein variant that is specifically defective in ATP hydrolysis (13).

Fig. 3.

EPR properties of NifEN in the presence of C₂H₂ and N₃⁻. (A and B) Temperature-dependency of the EPR spectrum of NifEN under turnover (A) and nonturnover (B) conditions of C₂H₂. (C and D) Temperature-dependency of the EPR spectrum of NifEN under turnover (C) and nonturnover (D) conditions of N₃⁻. Turnover samples (A and C) contain ATP, which is absent from nonturnover samples (B and D).

Fig. 4.

Interactions between C₂H₂, N₃⁻ and CO in NifEN-catalyzed reactions. (A and B) C₂H₂-reducing activities (A) and EPR properties (B) of NifEN with increasing N₃⁻ concentrations. Black, no N₃⁻; blue, 20 mM N₃⁻; green, 60 mM N₃⁻. (C and D) N₃⁻-reducing activities (C) and EPR properties (D) of NifEN with increasing C₂H₂ concentrations. Burgundy, no C₂H₂; pink, 10% C₂H₂; red, 60% C₂H₂. (E and F) C₂H₂-reducing activities (E) and EPR properties (F) of NifEN with increasing CO concentrations. Black, no CO; green, 20% CO. (G and H) N₃⁻-reducing activities (G) and EPR properties (H) of NifEN with increasing CO concentrations. Burgundy, no CO; red, 20% CO.

Fig. 5.

Plausible model of C₂H₂-and N₃⁻-binding to NifEN. N₃⁻ may have two binding sites at the active cofactor site of NifEN: one site only allows the binding of N₃⁻; whereas the other also allows the binding of C₂H₂. It is possible that, at high concentrations, N₃⁻ competes with C₂H₂ for binding at the shared site; and at low concentrations, N₃⁻ allosterically enhances the C₂H₂ reduction upon binding to the unshared site. Based on this model, C₂H₂ cannot inhibit N₃⁻ reduction completely, as the unshared site allows a certain level of N₃⁻ reduction to occur even when the same reaction is completely blocked by C₂H₂ at the shared site. Conversely, N₃⁻ can inhibit C₂H₂ reduction completely, as both sites can be occupied fully by N₃⁻.