Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases

Abstract

Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which facilitates the cleavage of the relatively inert triple bond of N2. Nitrogenase is most commonly associated with the molybdenum–iron cofactor called FeMoco or the M-cluster, and it has been the subject of extensive structural and spectroscopic characterization over the past 60 years. In the late 1980s and early 1990s, two “alternative nitrogenase” systems were discovered, isolated, and found to incorporate V or Fe in place of Mo. These systems are regulated by separate gene clusters; however, there is a high degree of structural and functional similarity between each nitrogenase. Limited studies with the V- and Fe-nitrogenases initially demonstrated that these enzymes were analogously active as the Mo-nitrogenase, but more recent investigations have found capabilities that are unique to the alternative systems. In this review, we will discuss the reactivity, biosynthetic, and mechanistic proposals for the alternative nitrogenases as well as their electronic and structural properties in comparison to the well-characterized Mo-dependent system. Studies over the past 10 years have been particularly fruitful, though key aspects about V- and Fe-nitrogenases remain unexplored.

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.…”

Special Issue on Nitrogenases and Homologous Systems

“…The V‐ and Fe‐only nitrogenases share high degrees of sequence and structural homology with the Mo‐nitrogenase . However, in addition to the ability to reduce N 2 to NH 3 , the alternative nitrogenases demonstrate the ability to reduce CO and/or CO 2 to various hydrocarbons at considerably higher efficiencies than their classic Mo counterpart . Although the physiological function of these reactions remain unclear, observation of the capability of nitrogenase to convert CO to hydrocarbons, such as C 3 H 8 and C 4 H 10 , is of significant importance, as it represents the first and only biological process to mirror the Fischer–Tropsch reaction that is used for the industrial production of synthetic fuel from CO.…”

Natural and Engineered Electron Transfer of Nitrogenase

“…Nitrogenase is a two-component metalloenzyme consisting of a reductase (iron or "Fe" protein) and a N 2 -reducing protein (MoFe protein), where the name "MoFe" refers to the metals employed in its catalytic cofactor. There are two alternative nitrogenases that are dependent on V (VFe) and Fe only (FeFe), although the Mo-nitrogenase system from the soil bacterium Azotobacter vinelandii will serve as the model to outline nitrogenase's mechanism ( Figure 1) [5]. The Fe protein of A. vinelandii is a homodimer of approximately 66 kDa in mass encoded by the nifH gene.…”

“…Thus, H 2 can be produced by unproductive or productive pathways, with increased H 2 evolution and MgATP hydrolysis resulting from a combination of both. 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].…”