Rethinking the Nitrogenase Mechanism: Activating the Active Site

Abstract

Metalloenzymes called nitrogenases (N₂ases) harness the reactivity of transition metals to reduce N₂ to NH₃. Specifically, N₂ases feature a multimetallic active site, called a cofactor, which binds and reduces N₂. The seven Fe centers and one additional metal center (Mo, V, or Fe) that make up the cofactor are all potential substrate binding sites. Unraveling the mechanism by which the cofactor binds N₂ and reduces N₂ to NH₃ represents a multifaceted challenge because cofactor activation is required for N₂ binding and functionalization to NH₃. Despite decades of fascinating contributions, the nature of N₂ binding to the active site and the structure of the activated cofactor remain unknown. Herein, we discuss the challenges associated with N₂ reduction and how transition metal complexes facilitate N₂ functionalization by coordinating N₂. We also review the activation and/or reaction mechanisms reported for small molecule catalysts and the Haber-Bosch catalyst and discuss their potential relevance to biological N₂ fixation. Finally, we survey what is known about the mechanism of N₂ase and highlight recent X-ray crystallographic studies supporting Fe-S bond cleavage at the active site to generate reactive Fe centers as a potential, underexplored route for cofactor activation. We propose that structural rearrangements, beyond electron and proton transfers, are key in generating the catalytically active state(s) of the cofactor. Understanding the mechanism of activation will be key to understanding N₂ binding and reduction.

Figure 1.. N₂ Coordination Modes Observed in Transition Metal Complexes

Dinitrogen binding at transition metal centers involves the metal center’s empty d-orbitals accepting electron density from one of the lone pairs of the N₂ ligand and π-back donation of the filled metal d-orbitals into the vacant π*-orbitals of the N₂ ligand (enclosed).^, Select examples of reported N₂ binding modes shown in complexes 1-4.^– For 2, Ar = 3,5-C₆H₃(CH₃)₂ and R = C(CD₃)₂CH₃.

Figure 2.. Structures of Select N₂ Fixation Catalysts

Chemdraw representation of the nitrogenase active site (FeMoco) and select examples of molecular N₂-to-NH₃ reduction catalysts 5-7.^–,

Figure 3.. Abbreviated Mechanisms for N₂-to-NH₃ Reduction Featuring N₂H_x Intermediates

Distal/Chatt (top), alternating (bottom), and hybrid (diagonal) pathways for N₂ reduction.^,, H₂ evolution from off-path M(H)₂ species shown in grey. M(H)₂ forms from unproductive M-H bond formation vs. N-H bond formation.^,,

Figure 4.. Simplified Reaction Mechanism for the Haber-Bosch Process

Rate-determining chemadsorption of N₂ forms surface bound N atoms which recombine with H atoms to yield NH₃. Green circles represent a highly simplified iron surface.

Figure 5.. N₂ Cleavage Across Multiple Iron Centers

An example of N₂ cleavage across multiple iron centers resulting in the formation of iron nitrides.

Figure 6.. Dissociative Mechanism for N₂ Reduction Involving Two Metal Centers

Generalized pathway for N₂-to-NH₃ fixation in which N-N bond cleavage occurs before N-H functionalization.

Figure 7.. Simplified MoFe Protein Cycle of the Lowe-Thorneley Kinetic Model

The various E states represent the different MoFe protein states as denoted by Lowe and Thorneley. Each arrow in the MoFe protein cycle indicates one Fe protein cycle. The enclosed activation steps represent chemical reactions required prior to N₂ binding, with N₂ binding and N₂-dependent H₂ evolution occurring at the E₃ or E₄ state and N₂-independent H₂ evolution at the E₂ state. NH₃ release occurs at E₅, E₆, and/or E₇.^,–

Figure 8.. Active Site Structures of Mo and V Nitrogenases with Different Ligands in the Belt Positions

Different ligand bound forms of FeMoco and FeVco with select hydrogen-bonding interactions shown in structures 10 and 13.^, For 11, Se can also populate the S5A or S3A positions depending on the reaction conditions. For 12 and 13, CO₃²⁻ bridges Fe4 and Fe5 of FeVco.^,