Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2

Abstract

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N₂ reduction relative to H₂ evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H₂ is a key feature in the N₂ reduction mechanism. The N₂ reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N₂ reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N₂ reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N₂ reduction (1 atm) of 181 ± 5 nmol NH₃ min^-1 mg^-1 FeFe protein, for proton reduction (in the absence of N₂) of 1085 ± 41 nmol H₂ min^-1 mg^-1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C₂H₄ min^-1 mg^-1 FeFe protein. Under turnover conditions, N₂ reduction is inhibited by H₂ and the enzyme catalyzes the formation of HD when presented with N₂ and D₂. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N₂ reduction occurring by reductive elimination of H₂.

Figure 1

General nitrogenase architecture, nitrogenase cofactors, and N₂ activation mechanism. (A) Schematic of nitrogenases showing the electron delivery component (Fe protein), catalytic component (MoFe, VFe, or FeFe protein), metal clusters, and electron delivery pathway. The differing metal atom (M in red) is Mo, V, or Fe. MoFe protein is an α₂β₂ heterotetramer, while VFe and FeFe proteins are α₂β₂γ₂ heterohexamers. (B) Structural models of nitrogenase cofactors with homocitrate on the right based on crystal structures for MoFe and VFe proteins. The carbonate in the FeV-cofactor has not been unambiguously assigned. No structure for the FeFe-cofactor is available, so a model is proposed based on available data. (C) Simplified form of the N₂ activation mechanism, with cartoons of the Fe 2, 3, 6, 7 face of FeMo-cofactor in the E₀, E₂, and E₄ states (designation from the Lowe–Thorneley kinetic model) showing hydrides and protons and showing both the re/oa equilibrium with loss of H₂ and binding/reduction of N₂ (going right) and the two steps of H₂ generation by hydride protonation that occur in the absence of N₂ (going left). The exact location of the bound hydrides and protons is proposed.

Figure 2

Specific activities for substrate reduction by Mo- and Fe-nitrogenases. Shown are the N₂ partial pressure dependence of NH₃ formation (left) and C₂H₂ on C₂H₄ formation (right) in Fe-nitrogenase (●) and Mo-nitrogenase (■). N₂ and C₂H₂ apparent K_m and V_max values were determined by a fit of the data to the Michaelis–Menten equation (lines). Mo-nitrogenase: apparent K_m N₂ = 0.13 ± 0.03 atm, V_max = 713 ± 19 nmol NH₃ min⁻¹ mg⁻¹ MoFe protein, and apparent K_m C₂H₂ = 0.009 ± 0.0005 atm, V_max = 1876 ± 20 nmol C₂H₄ min⁻¹ mg⁻¹ MoFe protein. Fe-nitrogenase: apparent K_m N₂ = 0.56 ± 0.06 atm, V_max = 286 ± 15 nmol NH₃ min⁻¹ mg⁻¹ FeFe protein, and apparent K_m C₂H₂ = 0.14 ± 0.01 atm, V_max = 450 ± 18 nmol C₂H₄ min⁻¹ mg⁻¹ FeFe protein. Data are the average of two independent experiments with error bars. Assays were performed as described in Materials and Methods.

Figure 3

Effect of increasing partial pressure of N₂ (P_N2) on N₂ (●) and H⁺ (■) reduction in Mo-nitrogenase (top) and Fe-nitrogenase (bottom). Data points are connected with a solid line as a guide. Data are the average of two independent experiments with error bars. Assays were performed as described in Materials and Methods.

Figure 4

Effect of increasing partial pressure of acetylene (P_C2H2) on acetylene (●) and H⁺ (■) reduction in Mo-nitrogenase (top) and Fe-nitrogenase (bottom). Data points are connected with a solid line as a guide. Data are the average of two independent experiments with error bars. Assays were performed as described in Materials and Methods.

Figure 5

H₂ inhibition of N₂ reduction. Shown is the specific activity for N₂ reduction as a function of the partial pressure of H₂ for Mo-nitrogenase (A) and Fe-nitrogenase (B). Data are the average of two independent experiments with error bars. Assays were performed as described in Materials in Methods. For Mo-nitrogenase, N₂ was held at 0.2 atm and H₂ was varied. For Fe-nitrogenase, N₂ was held at 0.6 atm and H₂ was varied.

Figure 6

HD formation by nitrogenase. Shown is one FeS face of FeMo-cofactor with a reversible re/oa equilibrium for N₂ or D₂ binding. The pathway to the left leads to the formation of 2 equiv of HD. The deutrides (green) are not solvent exchangeable, whereas the protons (red) are solvent exchangeable.

Figure 7

HD formation by Fe-nitrogenase. Volumes of produced H₂ (upper) and HD (lower) (1 atm, 295 K) in the headspace of turnover sample vials as a function of the ratio of N₂ and D₂ partial pressures (see Materials and Methods); error bars represent standard deviations.