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Review
. 2014 Apr 23;114(8):4041-62.
doi: 10.1021/cr400641x. Epub 2014 Jan 27.

Mechanism of nitrogen fixation by nitrogenase: the next stage

Brian M Hoffman  1 , Dmitriy Lukoyanov, Zhi-Yong Yang, Dennis R Dean, Lance C Seefeldt
Affiliations
Review

Mechanism of nitrogen fixation by nitrogenase: the next stage

Brian M Hoffman et al. Chem Rev. .
No abstract available

PubMed Disclaimer

Figures

Figure 1
Figure 1
Molybdenum nitrogenase. (A) One catalytic half of the Fe protein:MoFe protein complex with the Fe protein homodimer shown in tan, the MoFe protein α subunit in green, and the β subunit in cyan. (B) Space filling and stick models for the 4Fe–4S cluster (F), P-cluster (P), and FeMo-co (M). Made with Pymol and ChemDraw using PDB:2AFK.
Figure 2
Figure 2
FeMo-cofactor and the side chains of selected amino acid residues of the MoFe protein. Numbering of iron atoms is according to the structure PDB coordinate 2AFK. Iron is shown in rust, molybdenum in magenta, nitrogen in blue, sulfur in yellow, carbon in gray, and oxygen in red.
Figure 3
Figure 3
Simplified LT kinetic scheme that highlights correlated electron/proton delivery in eight steps. Although in the full LT scheme N2 binds at either the E3 or E4 levels, the pathway through E3 is de-emphasized here. LT also denotes the protons bound to FeMo-co (e.g., E1H1); for clarity we have omitted these protons in this scheme.
Figure 4
Figure 4
Depiction of E4 as containing two [Fe–H–Fe] moieties, emphasizing the essential role of this key “Janus intermediate”, which comes at the halfway point in the LT scheme, having accumulated four [e–/H+], and whose properties have implications for the first and second halves of the scheme. Janus image adapted from http://www.plotinus.com/janus_copy2.htm. Figure adapted with permission from ref (156). Copyright 2013 American Chemical Society.
Scheme 1
Scheme 1
Figure 5
Figure 5
Mockups of the “Janus” E4 intermediate in which the two bridging hydrides [Fe–H–Fe] revealed by ENDOR spectroscopy are built onto the resting-state crystal structure. These models of FeMo-co have Fe6 as a “vertex” for the two bridging hydrides to facilitate reductive elimination. The figure was generated using the coordinate file PDB:2AFK. Iron is shown in rust, molybdenum in magenta, sulfur in yellow, carbon in dark gray, and hydrogen in light gray.
Figure 6
Figure 6
Formulations of E1–E4 derived from consideration of E4 as containing two bound hydrides and two protons. (A) Assuming reduction of the core in n = 1, 3 states. (B) Alternative formulation of E1–E4 under the assumption of hydride formation at every stage, in which case the core is formally oxidized for En, n = 1, 3. Symbols: M represents FeMo-co core; superscripts are charge difference between core and that of resting-state (commonly denoted MN); the number of bound protons/hydrides are indicated. Adapted with permission from ref (156). Copyright 2013 American Chemical Society.
Figure 7
Figure 7
Formation and relaxation of E2. In-line: The “on-path” two-step, ATP-dependent addition of two H+/e– to MoFe protein to form E2. Off-line: Representation of the exergonic (free energy, +|ΔGh|) “off-path” relaxation of E2, liberating H2 and directly regenerating E0 without intervention of Fe protein, and of the energetically (free energy, +|ΔGh|) and kinetically forbidden reverse of this process; E0′ is a putative intermediate state that causes the reaction of E0 not to be the microscopic reverse of the release of H2 from E2 (see text).
Figure 8
Figure 8
Comparison of distal (D) and alternating (A) pathways for N2 hydrogenation, highlighting the stages that best distinguish them, most especially noting the different stages at which NH3(1) is released.
Figure 9
Figure 9
Comparison of 35 GHz ReMims pulsed 15N ENDOR spectra of intermediates trapped during turnover of the α-70Ala/α-195Gln MoFe protein with 15N2H4, 15N2H2, and 15NH=N—CH3 (denoted 15MD). Adapted with permission from ref (207). Copyright 2011 American Chemical Society.
Figure 10
Figure 10
2K Q-band CW EPR spectrum of α-70Val→Ala, α-195His→Gln MoFe protein in resting-state (S = 3/2) and trapped during turnover with 14N2H4. Kramers intermediate I and non-Kramers intermediate, H, are noted in the turnover spectrum. Adapted with permission from ref (219). Copyright 2012 National Academy of Sciences.
Figure 11
Figure 11
Three-pulse ESEEM traces after decay-baseline subtraction for NK intermediate H of α-70Val→Ala, α-195His→Gln MoFe protein trapped during turnover with 14NH=14NH, 14NH=14NCD3, 14NH2—14NH2, 15NH=15NH, 15NH=14NCH3. Adapted with permission from ref (219). Copyright 2012 National Academy of Sciences.
Figure 12
Figure 12
Integration of LT kinetic scheme with “prompt” (P) alternating (A) pathway for N2 reduction. The ? represents the product of N2 binding with H2 release, whose identity is discussed below. Also shown is how diazene and hydrazine join the N2 reduction pathway. Note: M denotes FeMo-co in its entirety, and substrate-derived species are drawn to indicate stoichiometry only, not mode of substrate binding. En states, n = even, are Kramers states; n = odd are non-Kramers. MN denotes resting-state FeMo-co. Individual charges on M and a substrate fragment, not shown, sum to the charge on resting FeMo-co. Adapted with permission from ref (156) with corrections based on the re mechanism for H2 loss upon N2 binding discussed below. Copyright 2013 American Chemical Society.
Figure 13
Figure 13
Visualization of hp and re mechanisms for H2 release upon N2 (blue) binding to E4. The following is shown: the Fe-2,3,6,7 face of resting FeMo-co; the structure of FeMo-co must distort in different stages of catalysis. The Fe that binds N2 is presumed to be Fe6, as indicated by studies of α-70Val variants; when bold, red, Fe6 is formally reduced by two equivalents (see text). The bridging hydrides of E4 (green) are positioned to share an Fe “vertex”, as suggested by re mechanism of H2 release upon N2 binding. Alternative binding modes for N2-derived species can be envisaged.
Chart 1
Chart 1
Figure 14
Figure 14
Reversal of hp and re mechanisms upon D2 binding. Details as in Figure 13. Bold arrows replace equilibrium arrows to emphasize the relaxation process.
Figure 15
Figure 15
Formation of deuterated acetylenes during turnover under N2/D2/C2H2 as predicted according to re mechanism. Cartoons again depict the Fe2,3,6,7 face of resting-state FeMo-co, with no attempt to incorporate likely structural modifications. Figure shows that the “reverse” of re mechanism through displacement of N2 by D2 produces, successively, E4(2D) and E2(D), further showing potential reaction channels for capture of E4(2D) and E2(D) intermediates with C2H2.
Figure 16
Figure 16
Schematic mechanism for reaction of C2H2 with E4(2D) and E2(D). (A) Formation of C2H2D2, which follows Scheme 15.20 of Hartwig: mi = migratory insertion; re = reductive elimination. In braces: Possible alternative reaction channel that leads to formation of C2H3D, ap = alkenyl protonolysis. (B) Schematic mechanism for formation of C2H3D from reaction of C2H2 with E2(D). (C) Illustration of possibility that C2H2 displaces D2 formed by reductive elimination of the E4(2D) deuterides, leading to direct formation of C2H4 without D incorporation.
Figure 17
Figure 17
Time-dependent formation of 13C2H3D and 13C2H2D2, catalyzed by nitrogenase reduction of 13C2H2. 13C2H3D determined by GC/MS monitoring of m/z = 31 for a reaction mixture containing 13C2H2 and including D2 and N2 (■), just D2 (x inside □), or H2 and N2 (□). Inset: 13C2H2D2, m/z = 32, formation starting with 13C2H2/D2/N2 (●), just D2 (◊), or H2/N2 (○). Partial pressures of 0.02 atm 13C2H2, 0.25 atm N2, and 0.7 atm H2/D2, where present. The molar ratio of Fe protein to MoFe protein was 2:1. All assays incubated at 30 °C. Adapted with permission from ref (157). Copyright 2013 National Academy of Sciences.
Figure 18
Figure 18
Deuterated ethylene formation as a function of N2 partial pressure. The partial pressure of C2H2 was 0.02 atm and D2 was 0.6 atm. The molar ratio of Fe protein to MoFe protein was 4:1. Assay conditions as in Figure 17. Adapted with permission from ref (157). Copyright 2013 National Academy of Sciences.
Figure 19
Figure 19
Proposed mechanism displaying structures of all intermediates in nitrogen fixation, inspired by the assumption of primacy of hydride chemistry associated with the Fe2,3,6,7 face of FeMo-co, and containing a formal description of the transformations that convert each stage to the subsequent one. In I the mechanism tentatively adopts and visualizes the view of En states n = 1–4 presented in Figure 6B; in II it visualizes bridging hydrides by analogy, without evidence for or against terminal hydrides for n = 5–7. Likewise, the structure of the N2H2 species as end-on bound diazene is suggestive, not definitive, etc. I and II are connected by the re mechanism, Figure 13, lower. Formal charges are included as useful to help guide the reader.
Figure 20
Figure 20
Models for the two alternative modes for N2 binding at Fe6 of FeMo-cofactor in the E4(N2) state, with two protons bound to two adjacent sulfides as in Figure 4: (A) endo mode; (B) exo mode. The side chains of selected amino acid residues are shown as sticks. The figure was generated in Pymol by building N2 onto the resting-state of FeMo-co using the coordinate file PDB:2AFK. Iron is shown in rust, molybdenum in magenta, sulfur in yellow, carbon in dark gray, hydrogen in light gray, nitrogen in blue, and oxygen in red.
See this image and copyright information in PMC

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