The vital role that nitrogenase plays in sustaining life is examined from a biomolecular perspective. Nitrogen fixation is shown to occur in lightning and the Haber-Bosch process.
Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N
→ 2NH
reduction involves the reductive elimination (
) of H
from two Fe-H-Fe bridging ...hydrides bound to the catalytic 7Fe-9S-Mo-C-homocitrate FeMo-cofactor (FeMo-co). These studies rationalize the Lowe-Thorneley kinetic scheme's proposal of mechanistically obligatory formation of one H
for each N
reduced. They also provide an overall framework for understanding the mechanism of nitrogen fixation by nitrogenase. However, they directly pose fundamental questions addressed computationally here. We here report an extensive computational investigation of the structure and energetics of possible nitrogenase intermediates using structural models for the active site with a broad range in complexity, while evaluating a diverse set of density functional theory flavors. (
) This shows that to prevent spurious disruption of FeMo-co having accumulated 4
/H
it is necessary to include: all residues (and water molecules) interacting directly with FeMo-co via specific H-bond interactions; nonspecific local electrostatic interactions; and steric confinement. (
) These calculations indicate an important role of sulfide hemilability in the overall conversion of
to a diazene-level intermediate. (
) Perhaps most importantly, they explain (
) how the enzyme mechanistically couples exothermic H
formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutral
/oxidative-addition equilibrium, (
) while preventing the "futile" generation of two H
without N
reduction: hydride
generates an H
complex, but H
is only lost when displaced by N
, to form an end-on N
complex that proceeds to a diazene-level intermediate.
Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N₂ → 2NH₃ reduction involves the reductive elimination (re) of H₂ from two Fe–H–Fe ...bridging hydrides bound to the catalytic 7Fe–9S–Mo–C–homocitrate FeMo-cofactor (FeMo-co). These studies rationalize the Lowe–Thorneley kinetic scheme’s proposal of mechanistically obligatory formation of one H₂ for each N₂ reduced. They also provide an overall framework for understanding the mechanism of nitrogen fixation by nitrogenase. However, they directly pose fundamental questions addressed computationally here. We here report an extensive computational investigation of the structure and energetics of possible nitrogenase intermediates using structural models for the active site with a broad range in complexity, while evaluating a diverse set of density functional theory flavors. (i) This shows that to prevent spurious disruption of FeMo-co having accumulated 4e⁻/H⁺ it is necessary to include: all residues (and water molecules) interacting directly with FeMo-co via specific H-bond interactions; nonspecific local electrostatic interactions; and steric confinement. (ii) These calculations indicate an important role of sulfide hemilability in the overall conversion of E₀ to a diazene-level intermediate. (iii) Perhaps most importantly, they explain (iiia) how the enzyme mechanistically couples exothermic H₂ formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutral re/oxidative-addition equilibrium, (iiib) while preventing the “futile” generation of two H₂ without N₂ reduction: hydride re generates an H₂ complex, but H₂ is only lost when displaced by N₂, to form an end-on N₂ complex that proceeds to a diazene-level intermediate.
Bacteria that oxidize methane to methanol are central to mitigating emissions of methane, a potent greenhouse gas. The nature of the copper active site in the primary metabolic enzyme of these ...bacteria, particulate methane monooxygenase (pMMO), has been controversial owing to seemingly contradictory biochemical, spectroscopic, and crystallographic results. We present biochemical and electron paramagnetic resonance spectroscopic characterization most consistent with two monocopper sites within pMMO: one in the soluble PmoB subunit at the previously assigned active site (Cu
) and one ~2 nanometers away in the membrane-bound PmoC subunit (Cu
). On the basis of these results, we propose that a monocopper site is able to catalyze methane oxidation in pMMO.
Mechanism of Mo-dependent nitrogenase Seefeldt, Lance C; Hoffman, Brian M; Dean, Dennis R
Annual review of biochemistry,
01/2009, Letnik:
78
Journal Article
Recenzirano
Odprti dostop
Nitrogen-fixing bacteria catalyze the reduction of dinitrogen (N(2)) to two ammonia molecules (NH(3)), the major contribution of fixed nitrogen to the biogeochemical nitrogen cycle. The most widely ...studied nitrogenase is the molybdenum (Mo)-dependent enzyme. The reduction of N(2) by this enzyme involves the transient interaction of two component proteins, designated the iron (Fe) protein and the MoFe protein, and minimally requires 16 magnesium ATP (MgATP), eight protons, and eight electrons. The current state of knowledge on how these proteins and small molecules together effect the reduction of N(2) to ammonia is reviewed. Included is a summary of the roles of the Fe protein and MgATP hydrolysis, information on the roles of the two metal clusters contained in the MoFe protein in catalysis, insights gained from recent success in trapping substrates and inhibitors at the active-site metal cluster FeMo cofactor, and finally, considerations of the mechanism of N(2) reduction catalyzed by nitrogenase.
Reduction of Substrates by Nitrogenases Seefeldt, Lance C; Yang, Zhi-Yong; Lukoyanov, Dmitriy A ...
Chemical reviews,
06/2020, Letnik:
120, Številka:
12
Journal Article
Recenzirano
Odprti dostop
Nitrogenase is the enzyme that catalyzes biological N2 reduction to NH3. This enzyme achieves an impressive rate enhancement over the uncatalyzed reaction. Given the high demand for N2 fixation to ...support food and chemical production and the heavy reliance of the industrial Haber–Bosch nitrogen fixation reaction on fossil fuels, there is a strong need to elucidate how nitrogenase achieves this difficult reaction under benign conditions as a means of informing the design of next generation synthetic catalysts. This Review summarizes recent progress in addressing how nitrogenase catalyzes the reduction of an array of substrates. New insights into the mechanism of N2 and proton reduction are first considered. This is followed by a summary of recent gains in understanding the reduction of a number of other nitrogenous compounds not considered to be physiological substrates. Progress in understanding the reduction of a wide range of C-based substrates, including CO and CO2, is also discussed, and remaining challenges in understanding nitrogenase substrate reduction are considered.
The ability of certain transition metals to mediate the reduction of N2 to NH3 has attracted broad interest in the biological and inorganic chemistry communities. Early transition metals such as Mo ...and W readily bind N2 and mediate its protonation at one or more N atoms to furnish M(N x H y ) species that can be characterized and, in turn, extrude NH3. By contrast, the direct protonation of Fe–N2 species to Fe(N x H y ) products that can be characterized has been elusive. Herein, we show that addition of acid at low temperature to (TPB)Fe(N2)Na(12-crown-4) results in a new S = 1/2 Fe species. EPR, ENDOR, Mössbauer, and EXAFS analysis, coupled with a DFT study, unequivocally assign this new species as (TPB)FeN–NH2+, a doubly protonated hydrazido(2−) complex featuring an Fe-to-N triple bond. This unstable species offers strong evidence that the first steps in Fe-mediated nitrogen reduction by (TPB)Fe(N2)Na(12-crown-4) can proceed along a distal or “Chatt-type” pathway. A brief discussion of whether subsequent catalytic steps may involve early or late stage cleavage of the N–N bond, as would be found in limiting distal or alternating mechanisms, respectively, is also provided.
We have shown that the key state in N2 reduction to two NH3 molecules by the enzyme nitrogenase is E4(4H), the “Janus” intermediate, which has accumulated four e–/H+ and is poised to undergo ...reductive elimination of H2 coupled to N2 binding and activation. Initial 1H and 95Mo ENDOR studies of freeze-trapped E4(4H) revealed that the catalytic multimetallic cluster (FeMo-co) binds two Fe-bridging hydrides, Fe–H–Fe. However, the analysis failed to provide a satisfactory picture of the relative spatial relationships of the two Fe–H–Fe. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted as E4(4H)(a), with two parallel Fe–H–Fe planes bridging pairs of “anchor” Fe on the Fe2,3,6,7 face of FeMo-co. However, the relative energies of structures E4(4H)(b), with one bridging and one terminal hydride, and E4(4H)(c), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties in the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E4(4H)(c), and additional structures have been proposed in the DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic 1H-ENDOR experiments using improved instrumentation, Mims 2H ENDOR, and a recently developed pulsed-ENDOR protocol (“PESTRE”) to obtain absolute hyperfine interaction signs. These measurements are coupled to DFT structural models through an analytical point-dipole Hamiltonian for the hydride electron–nuclear dipolar coupling to its “anchoring” Fe ions, an approach that overcomes limitations inherent in both experimental interpretation and computational accuracy. The result is the freeze-trapped, lowest-energy Janus intermediate structure, E4(4H)(a).
Nitrogenase: A Draft Mechanism Hoffman, Brian M; Lukoyanov, Dmitriy; Dean, Dennis R ...
Accounts of chemical research,
02/2013, Letnik:
46, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Biological nitrogen fixation, the reduction of N2 to two NH3 molecules, supports more than half the human population. The predominant form of the enzyme nitrogenase, which catalyzes this reaction, ...comprises an electron-delivery Fe protein and a catalytic MoFe protein. Although nitrogenase has been studied extensively, the catalytic mechanism has remained unknown. At a minimum, a mechanism must identify and characterize each intermediate formed during catalysis and embed these intermediates within a kinetic framework that explains their dynamic interconversion. The Lowe–Thorneley (LT) model describes nitrogenase kinetics and provides rate constants for transformations among intermediates (denoted E n , where n is the number of electrons (and protons), that have accumulated within the MoFe protein). Until recently, however, research on purified nitrogenase had not characterized any E n state beyond E0. In this Account, we summarize the recent characterization of three freeze-trapped intermediate states formed during nitrogenase catalysis and place them within the LT kinetic scheme. First we discuss the key E4 state, which is primed for N2 binding and reduction and which we refer to as the “Janus intermediate” because it lies halfway through the reaction cycle. This state has accumulated four reducing equivalents stored as two Fe–H–Fe bridging hydrides bound to the active-site iron–molybdenum cofactor (7Fe–9S–Mo–C–homocitrate; FeMo-co) at its resting oxidation level. The other two trapped intermediates contain reduced forms of N2. One, intermediate, designated I , has S = 1/2 FeMo-co. Electron nuclear double resonance/hyperfine sublevel correlation (ENDOR/HYSCORE) measurements indicate that I is the final catalytic state, E8, with NH3 product bound to FeMo-co at its resting redox level. The other characterized intermediate, designated H , has integer-spin FeMo-co (non-Kramers; S ≥ 2). Electron spin echo envelope modulation (ESEEM) measurements indicate that H contains the −NH2 fragment bound to FeMo-co and therefore corresponds to E7. These assignments in the context of previous studies imply a pathway in which (i) N2 binds at E4 with liberation of H2, (ii) N2 is promptly reduced to N2H2, (iii) the two N’s are reduced in two steps to form hydrazine-bound FeMo-co, and (iv) two NH3 are liberated in two further steps of reduction. This proposal identifies nitrogenase as following a “prompt-alternating ( P-A )” reaction pathway and unifies the catalytic pathway with the LT kinetic framework. However, the proposal does not incorporate one of the most puzzling aspects of nitrogenase catalysis: obligatory generation of H2 upon N2 binding that apparently “wastes” two reducing equivalents and thus 25% of the total energy supplied by the hydrolysis of ATP. Because E 4 stores its four accumulated reducing equivalents as two bridging hydrides, we propose an answer to this puzzle based on the organometallic chemistry of hydrides and dihydrogen. We propose that H2 release upon N2 binding involves reductive elimination of two hydrides to yield N2 bound to doubly reduced FeMo-co. Delivery of the two available electrons and two activating protons yields cofactor-bound diazene, in agreement with the P-A scheme. This keystone completes a draft mechanism for nitrogenase that both organizes the vast body of data on which it is founded and serves as a basis for future experiments.
Conspectus The seeds for recognition of the vast superfamily of radical S-adenosyl-l-methionine (SAM) enzymes were sown in the 1960s, when Joachim Knappe found that the dissimilation of pyruvate was ...dependent on SAM and Fe(II), and Barker and co-workers made similar observations for lysine 2,3-aminomutase. These intriguing observations, coupled with the evidence that SAM and Fe were cofactors in radical catalysis by these enzyme systems, drew us in the 1990s to explore how Fe(II) and SAM initiate radical reactions. Our early work focused on the same enzyme Knappe had originally characterized: the pyruvate formate-lyase activating enzyme (PFL-AE). Our discovery of an iron–sulfur cluster in this enzyme, together with similar findings for other SAM-dependent enzymes at the time, led to the recognition of an emerging class of enzymes that use iron–sulfur clusters to cleave SAM, liberating the 5′-deoxyadenosyl radical (5′-dAdo•) that initiates radical reactions. A major bioinformatics study by Heidi Sofia and co-workers identified the enzyme superfamily denoted Radical SAM, now known to span all kingdoms of life with more than 100,000 unique sequences encoding enzymes that catalyze remarkably diverse reactions. Despite the limited sequence similarity and vastly divergent reactions catalyzed, the radical SAM enzymes appear to employ a common mechanism for initiation of radical chemistry, a mechanism we have helped to clarify over the last 25 years. A reduced 4Fe-4S+ cluster provides the electron needed for the reductive cleavage of SAM. The resulting 4Fe-4S2+ cluster can be rereduced either by an external reductant, with SAM acting as a cosubstrate, or by an electron provided during the reformation of SAM in cases where SAM is used as a cofactor. The amino and carboxylate groups of SAM bind to the unique iron of the catalytic 4Fe-4S cluster, placing the sulfonium of SAM in close proximity to the cluster. Surprising recent results have shown that the initiating enzymatic cleavage of SAM generates an organometallic intermediate prior to liberation of 5′-dAdo•, which initiates radical chemistry on the substrate. This organometallic intermediate, denoted Ω, has a 5′-deoxyadenosyl moiety directly bound to the unique iron of the 4Fe-4S cluster via the 5′-C, giving a structure that is directly analogous to the Co-(5′-C) bond of the organometallic cofactor adenosylcobalamin. Our observation that this intermediate Ω is formed throughout the superfamily suggests that this is a key intermediate in initiating radical SAM reactions, and that organometallic chemistry is much more broadly relevant in biology than previously thought.
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