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The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V 3+ , 3Fe 3+ , 4Fe 2+ ]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin ( S = 1, 2 etc. ). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined 51 V ( I = 7/2) hyperfine splitting, a iso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state)more »with metal-ion valencies, [1V 3+ , 4Fe 3+ , 3Fe 2+ ]. Our findings suggest that the V 3+ does not change valency throughout the catalytic cycle.« less

Kang et al . (Reports, 19 June 2020, p. 1381) report a structure of the nitrogenase MoFe protein that is interpreted to indicate binding of N 2 or an N 2 -derived species to the active-site FeMo cofactor. Independent refinement of the structure and consideration of biochemical evidence do not support this claim.

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.

Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N₂→ 2NH₃reduction involves the reductive elimination (re) of H₂from 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 H₂for each N₂reduced. 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 4[e⁻/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 ofE₀to a diazene-level intermediate. (iii) Perhaps most importantly, they explain (iiia) how the enzyme mechanistically couples exothermic H₂formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutralre/oxidative-addition equilibrium, (iiib) while preventing the “futile”more »generation of two H₂without N₂reduction: hydrideregenerates an H₂complex, but H₂is only lost when displaced by N₂, to form an end-on N₂complex that proceeds to a diazene-level intermediate.

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