Two ligand-binding sites in CO-reducing V nitrogenase reveal a general mechanistic principle

Abstract

Besides its role in biological nitrogen fixation, vanadium-containing nitrogenase also reduces carbon monoxide (CO) to hydrocarbons, in analogy to the industrial Fischer-Tropsch process. The protein yields 93% of ethylene (C₂H₄), implying a C-C coupling step that mandates the simultaneous binding of two CO at the active site FeV cofactor. Spectroscopic data indicated multiple CO binding events, but structural analyses of Mo and V nitrogenase only confirmed a single site. Here, we report the structure of a two CO-bound state of V nitrogenase at 1.05 Å resolution, with one μ-bridging and one terminal CO molecule. This additional, specific ligand binding site suggests a mechanistic route for CO reduction and hydrocarbon formation, as well as a second access pathway for protons required during the reaction. Moreover, carbonyls are strong-field ligands that are chemically similar to mechanistically relevant hydrides that may be formed and used in a fully analogous fashion.

Fig. 1. The active site cofactor of the two structurally characterized nitrogenases.

(A) FeMo cofactor in the resting state E₀ [extended data, Fig. 5; Protein Data Bank (PDB) 3U7Q] (62). (B) FeV cofactor in the resting state (PDB 5N6Y) (6). (C) FeMo cofactor in the CO-inhibited state (PDB 4TVK) (21). (D) FeV cofactor in the low-CO state (PDB 7ADR) (24). All atoms of the cofactors are labeled according to standard convention.

Fig. 2. The high-CO state of V nitrogenase.

(A) Stereo image showing a 2F_o–F_c difference electron density map in the vicinity of FeV cofactor, contoured at the 2σ level. The μ₂-bridging μCO ligand is modeled at full occupancy q, the terminal tCO ligand at Fe6 is modeled with q = 0.5. (B) Detail of the bound tCO molecule at Fe6 (stereo image). The electron density map is contoured at 1σ (gray), 2σ (blue), 3σ (orange), and 3.5σ (red) to highlight the distinct maxima for the C and O atoms and their different electron density peak heights. Distances are given in Ångstrøm (10⁻¹⁰ m).

Fig. 3. Binding of the second CO ligand.

(A) Bond distances around the μ₂-bridging CO ligand in the low-CO state of V nitrogenase. (B) Changes in bond distances and angles upon binding of the second CO ligand, as a terminal carbonyl to Fe6. Note that due to the 7° rotation of μCO, the hydrogen bond from its oxygen atom to residue H180 contracts from 2.87 to 2.74 Å. (C) Bond distances in the high-CO state of V nitrogenase. (D) IR difference spectra for ¹²CO and ¹³CO versus N₂-pressurized VFe protein. The distinct signals are in a typical range for terminal carbonyls and show an isotope shift of 43 cm⁻¹. (E) Kinetics of ¹²tCO binding (above) and dissociation (below). (F) The time course for (D) shows not only rapid binding but also rapid dissociation of CO from the tCO site.

Fig. 4. Mechanistic implications for the different reactivities of nitrogenases.

Nitrogenase catalysis according to the kinetic scheme of Thorneley and Lowe (extended data, Fig. 5) proceeds along successive e⁻/H⁺ transfer steps from the resting state E₀ (1) to a hydride-bridged state E₂ (2). Here, CO can bind to the cofactor, and we propose an initial terminal binding to Fe6 that is in equilibrium with a bridging carbonyl and a terminal hydride (3). The protonation of the unstable terminal hydride leads to release of H₂, with the μ₂-bridging carbonyl remaining in a formal E₀ state that was characterized structurally and spectroscopically as “low-CO” state (4). Alternatively, productive CO reduction by VFe protein may be initiated by an insertion reaction to yield a formyl adduct as a first step (5), while in this work, we show that pressurization of (4) leads to the “high-CO” state (6) that reveals Fe6 as the site for the terminal binding of ligands. While the two CO-bound states (4) and (6) likely are not on path for CO reduction, the concurrent bridging and terminal binding of two ligands rationalizes the mechanistically critical step (3) and also provides a new outlook on the initial steps of N₂ activation. These require the enzyme to reach the E₄ state that holds two hydrides. If one of these is bridging and the other terminal (7) in analogy to the high-CO state (6), then terminal N₂ binding to Fe6 could force a shift of the terminal hydride to become bridging, triggering the reductive elimination (re) of H₂ only after the substrate is in position (8) and leaving the enzyme in a super-reduced state that transfers two electrons to N₂, effectively breaking the stable triple bond of dinitrogen (9). The schemes depict a fragment of the cofactor including Fe sites Fe2 and Fe6 and the interstitial carbide (C).

Fig. 5. Implications for N₂ reduction by nitrogenase.

(A) Hypothetical E₄ state with hydrides at the μ- and the t-site. Although highly energetic, the state is stabilized by Q176 interacting with the μH. (B) Schematic view of FeV co with the μ- and t-sites highlighted. While H180 serves as proton donor for intermediates bound to the μ-site or Fe2, the t-site is likely supplied with H⁺ from the hydroxyl group of homocitrate. (C) The mechanistic suggestion of a doubly dihydride-bridged cofactor in the E₄ state [Fig. 4, (8)] is not without precedent. (D) The N₂-bound diiron complex (Fe₂(μ-H)₂-N₂)[SiP₂O] reported by Rittle and Peters (55) affords a highly similar topology. The compound was stable at room temperature and reversibly bound a second terminal N₂ at the other Fe site upon cooling to 193 K. Reduction to a mixed-valent Fe^II:Fe^I state led to a 10⁶-fold rate enhancement of N₂ binding. (E) The crystal structure of (D) reveals that the μ₂-hydrides are juxtaposed, likely adding to their stability. In contrast, nitrogenase cofactors are more rigid due to the remaining belt ligands, possibly imposing a Fe-Fe distance and geometry that favors the reductive elimination of H₂, as soon as the binding of N₂ displaces the tH to a bridging position.