Reduction of N by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear. Here we report the structural analysis of vanadium nitrogenase with a bound intermediate, interpreted as a μ-bridging, protonated nitrogen that implies the site and mode of substrate binding to the cofactor. Binding results in a flip of amino acid glutamine 176, which hydrogen-bonds the ligand and creates a holding position for the displaced sulfide. The intermediate likely represents state E or E of the Thorneley-Lowe model and provides clues to the remainder of the catalytic cycle.
Nitrogenases catalyze the reduction of N2 gas to ammonium at a complex heterometallic cofactor. Most commonly this is the FeMo cofactor (FeMoco), a [Mo:7Fe:9S:C] cluster whose exact reactivity and substrate binding mode remain unknown. Alternative nitrogenases replace molybdenum with either vanadium or iron and differ in reactivity, prominently in the ability of vanadium nitrogenase to reduce CO to hydrocarbons. Here we report the 1.35 Å structure of vanadium nitrogenase from Azotobacter vinelandii. The 240 kDa protein contains an additional α-helical subunit not present in molybdenum nitrogenase. The FeV cofactor (FeVco) is a [V:7Fe:8S:C] cluster with a homocitrate ligand to vanadium. Unexpectedly, it lacks one sulfide ion compared to FeMoco that is replaced by a bridging ligand, likely a μ-1,3-carbonate. The anion fits into a pocket within the protein that is obstructed in molybdenum nitrogenase, and its different chemical character helps to rationalize the altered chemical properties of this unique N2- and CO-fixing enzyme.
The [Mo:7Fe:9S:C] iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation. Only recently its atomic structure was clarified, while its reactivity and electronic structure remain under debate. Here we show that for its resting S=3/2 state the common iron oxidation state assignments must be reconsidered. By a spatially resolved refinement of the anomalous scattering contributions of the 7 Fe atoms of FeMoco, we conclude that three irons (Fe1/3/7) are more reduced than the other four (Fe2/4/5/6). Our data are in agreement with the recently revised oxidation state assignment for the molybdenum ion, providing the first spatially resolved picture of the resting-state electron distribution within FeMoco. This might provide the long-sought experimental basis for a generally accepted theoretical description of the cluster that is in line with available spectroscopic and functional data.
High-resolution X-ray spectroscopy provides insights into the electronic structural differences between the nitrogenase FeMoco and FeVco clusters.
The reaction catalyzed by the nitrogenase enzyme involves breaking the stable triple bond of the dinitrogen molecule and is consequently considered among the most challenging reactions in biology. While many aspects regarding its atomic mechanism remain to be elucidated, a kinetic scheme established by David Lowe and Roger Thorneley has remained a gold standard for functional studies of the enzyme for more than 30 years. Recent three-dimensional structures of ligand-bound states of molybdenum- and vanadium-dependent nitrogenases have revealed the actual site of substrate binding on the large active site cofactors of this class of enzymes. The binding mode of an inhibitor and a reaction intermediate further substantiate a hypothesis by Seefeldt, Hoffman, and Dean that the activation of N is made possible by a reductive elimination of H that leaves the cofactor in a super-reduced state that can bind and reduce the inert N molecule. Here we discuss the immediate implications of the structurally observed mode of binding of small molecules to the enzyme with respect to the early stages of the Thorneley-Lowe mechanism of nitrogenase. Four consecutive single-electron reductions give rise to two bridging hydrides at the cluster surface that can recombine to eliminate H and enable the reduced cluster to bind its substrate in a bridging mode.
The first direct evidence is provided for the presence of an interstitial carbide in the Fe–V cofactor of Azotobacter vinelandii vanadium nitrogenase. As for our identification of the central carbide in the Fe–Mo cofactor, we employed Fe Kβ valence-to-core X-ray emission spectroscopy and density functional theory calculations, and herein report the highly similar spectra of both variants of the cofactor-containing protein. The identification of an analogous carbide, and thus an atomically homologous active site in vanadium nitrogenase, highlights the importance and influence of both the interstitial carbide and the identity of the heteroatom on the electronic structure and catalytic activity of the enzyme.
A molybdenum L-edge X-ray absorption spectroscopy (XAS) study is presented for native and oxidized MoFe protein of nitrogenase as well as Mo-Fe model compounds. Recently collected data on MoFe protein (in oxidized and reduced forms) is compared to previously published Mo XAS data on the isolated FeMo cofactor in NMF solution and put in context of the recent Mo K-edge XAS study, which showed a MoIII assignment for the molybdenum atom in FeMoco. The L3-edge data are interpreted within a simple ligand-field model, from which a time-dependent density functional theory (TDDFT) approach is proposed as a way to provide further insights into the analysis of the molybdenum L3-edges. The calculated results reproduce well the relative spectral trends that are observed experimentally. Ultimately, these results give further support for the MoIII assignment in protein-bound FeMoco, as well as isolated FeMoco.
The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfDKG heterohexamer.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.