Rethinking the Nitrogenase Mechanism: Activating the Active Site

Abstract

Metalloenzymes called nitrogenases (N 2 ases) harness the reactivity of transition metals to reduce N 2 to NH 3. Specifically, N 2 ases feature a multimetallic active site, called a cofactor, which binds and reduces N 2. 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 N 2 and reduces N 2 to NH 3 represents a multifaceted challenge because cofactor activation is required for N 2 binding and functionalization to NH 3. Despite decades of fascinating contributions, the nature of N 2 binding to the active site and the structure of the activated cofactor remain unknown. Herein, we discuss the challenges associated with N 2 reduction and how transition-metal complexes facilitate N 2 functionalization by coordinating N 2. 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 N 2 fixation. Finally, we survey what is known about the mechanism of N 2 ase 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 these processes will be key to unveiling the mechanism of N 2 binding and reduction.

Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

“…It is also possible that proton tunneling, which is generally thought to have a very large KIE (but also see references 96 and 97 ) and has been proposed to occur in nitrogenase ( 79 ), could be contributing to the KIE observed here, although we note that the temperature effect observed here is opposite the predicted effect for tunneling ( 80 , 81 ). Computational models, which can distinguish the rates of hydrogenation based on 1 H and 2 H, and might be able to shed light on the mechanism responsible for the observed fractionation and whether the currently proposed, multistep mechanisms of hydrogenation by nitrogenase ( 82 – 85 ) are compatible with the measured KIE of 2.1. The clumped isotopic composition of methane produced by nitrogenase could also provide additional constraints.…”

“…Computational models can distinguish the rates of hydrogenation based on 1 H and 2 H and might be able to shed light on the mechanism responsible for the observed fractionation and whether the currently proposed, multi-step mechanisms of hydrogenation by nitrogenase (85)(86)(87)(88) are compatible with the measured KIE of ~2. The clumped isotopic composition of methane produced by nitrogenase could also provide additional constraints.…”

Natural and Engineered Electron Transfer of Nitrogenase

“…Notably, the fixation of each N 2 also results in the evolution of at least one equivalent of H 2 . Lowe and Thorneley developed an early model to describe the observation of H 2 formation, which has since developed into a model that highlights the pivotal nature of a 4e − -reduced FeMo-co known as the E 4 state [8,10,[21][22][23]. By this model, the resting FeMo-co in its E 0 state accumulates individual electrons and protons during each Fe protein association event in order to reach the E 4 state at which N 2 binds and undergoes subsequent reduction.…”

“…Questions therefore remain surrounding the reversibility or catalytic bias of the E 4 state, given that the alternative nitrogenases also appear to follow the same re mechanism while appearing to be less-efficient at N 2 fixation [5,25]. A "just-in-time" mechanism could serve as a useful model to justify the rate-limiting nature of electron transfer from the Fe protein (~13 s −1 ) such that unproductive H 2 formation and the oa of H 2 is minimized [23]. Finally, research has also questioned the suitability of the proposed re/oa model for N 2 fixation by nitrogenase [26].…”

Chimeric Interaction of Nitrogenase‐Like Reductases with the MoFe Protein of Nitrogenase

“…Tremendous progress in understanding biological dinitrogen fixation was made in recent years . Among other things, the key steps for the maturation of the sophisticated metallocenters have been deciphered, which might be an important step towards the design of plants or plant‐associated organisms with improved nitrogen‐fixing ability.…”