High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E4(4H)

Abstract

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).

Figure 1.

Crystal structure of FeMo-co. Fe is show in rust, Mo in cyan, S in yellow, carbide in dark grey, carbon in light gray, N in blue and O in red. The Fe atoms of catalytic 4Fe-4S face are labelled as 2, 3, 6, and 7. The α−275^Cys and α−442^His FeMo-co ligands along with two amino acids, α−70^Val and α−195^His, that approach the FeMo-co are also shown. The image was created using PDB coordinate 2AFI.

Figure 2.

Simplified kinetic scheme for nitrogen reduction that focuses on the electron-accumulation and FeMo-co activation stages. E_n notation: n = number of [e⁻/H⁺] added to resting-state FeMo-co; in parentheses, stoichiometry of H/N bound to FeMo-co.

Fig. 3:

Lowest-energy structures of the Janus intermediate as seen in recent density functional theory computations E₄(4H)^(a,b,c), plus a high-energy structure with singly protonated carbide that has been proposed based on other computations, E₄(3H;CH)^– and the P-7 structure proposed by Siegbahn,^, which results from the accumulation of 7 [e-/H+]. The relative electronic energies (ΔE in kJ/mol calculated at DFT/BP86 level) for E₄(4H)^(a,b,c) and E₄(3H;CH) along with selected Fe-H distances (in Angstrom) from Ref. 13 are provided. Fe-H distances for P-7 from Ref. 17 are also given. Note that the Fe-H distances of a [Fe-H-Fe] are not typically equal. Irons are shown in rust, molybdenum in cyan, carbon in gray, sulfurs in yellow, protic hydrogens in green, and hydrides in red.

Fig. 4.

35 GHz ¹H stochastic-field modulation detected (stochastic CW) ENDOR spectra of E₄(4H) acquired at g = 1.991 and 1.964 (Black); simulations generated as described below (Red). The signal with ν₀(¹H) ± ~3MHz, with both positive and negative features, represents transient responses from weakly coupled, more distant protons. Experimental Conditions: Microwave frequency, 35.045 GHz; modulation amplitude, 2.7 G; microwave power, 1 μW; RF power, 10 W; temperature, 2 K; stochastic sequence (sample/delay/RF), 1/3/3 ms; the bandwidth of the RF excitation was broadened to 100 kHz.

Fig 5.

A) Single-crystal-like ENDOR spectra collected at g₁ and g₃ (slight splittings in H1 peaks at g₁ indicate the rigorously single-crystal limit has not been reached). The figure illustrates the two possible scenarios by which the hydride signals can evolve across the EPR envelope in a 2D field-frequency pattern of ENDOR spectra collected from g₁ to g₃. In the first scenario, the patterns of H1 and H2 do not ‘cross’ at intermediate fields, i.e. A(H1) > A(H2) at g₁ and A(H1) > A(H2) at g₃ (solid lines). In the second scenario, the patterns of H1 and H2 do ‘cross’ at intermediate fields, so that A(H1) > A(H2) at g₁ but A(H1) < A(H2) at g₃ (dashed lines). B) Alternative models for hydride binding to FeMo-co, where a 4Fe4S face is depicted as an oval. Both hydrides may be terminal (model denoted T/T) or bridging (B/B), or the intermediate may contain one terminal and one bridging hydride, denoted T/B or B/T depending on the assignment to H1 and H2 (compare part A of this Figure). For each of these four models, either of the two ‘crossing’ scenarios illustrated in A can obtain, leading to a total of 8 possible bases for the 2D field-frequency plot.

Fig. 6.

Full 35 GHz ¹H stochastic CW field-frequency ENDOR pattern for E₄(4H). Experimental data (black), and sum of simulations for ¹H1 and ¹H2 (red), normalized at each g value. Curves running across the 2D plot were added as a guide to the eye, connecting the outer edges of ¹H1 (blue) and ¹H2 (green) spectral features along the different g values. Simulation parameters (B/B model) are listed in Table 1. At selected g values, a complementary ¹H field-swept CW or ²H Mims ENDOR spectrum (thinner line; ²H spectra were collected on D₂O turnover samples and scaled to ¹H frequencies by factor g_N(¹H)/g_N(²H) = 6.5) is shown to illustrate the match of experiment and simulation in the low-frequency region; for further ²H measurements, Fig S12. Experimental Conditions: ¹H stochastic CW ENDOR: same as in Fig. 4. ¹H field-swept CW ENDOR: Microwave frequency, 35.036 GHz; modulation amplitude, 0.7 G; microwave power, 1 μW; RF power, 10 W; RF scan speed, 1 MHz/s; temperature, 2 K; the bandwidth of the RF excitation was broadened to 100 kHz. ²H Mims ENDOR: 34.890 GHz; π/2 = 50 ns; τ = 400 ns; t_RF = 30 μs; repetition time, 50 ms; temperature, 2 K.

Fig. 7.

Absolute hyperfine sign determination for hydride H1 by PESTRE at g = 1.977. A) 35 GHz ¹H stochastic CW (thicker black line) and Davies (thinner black line) ENDOR responses around the ν₊ branch of the ¹H1 doublet, and sum (red) and separate ¹H1 (blue), ¹H2 (green) simulations. Simulation parameters (B/B model) are listed in Table 1. B) ¹H PESTRE trace collected from the ν₊ branch of the ¹H1 doublet. Experimental Conditions: A) ¹H stochastic CW ENDOR: same as in Fig. 4. ¹H Davies ENDOR: Microwave frequency, 34.776 GHz; π = 60 ns; τ = 450 ns; t_RF = 35 μs; repetition time, 100 ms; t_mix = 5 μs; temperature, 2 K. B) Same as in A (¹H Davies) except for: repetition time, 200 ms; RF frequency, 65.5 MHz.

Fig. 8.

Absolute hyperfine sign determination for hydride H2 by PESTRE at g₂ = 2.008. A) 35 GHz ¹H stochastic CW (thicker black line) and Davies (thinner black line) ENDOR responses around the ν_- (black arrow) and ν₊ (purple arrow) branch of the ¹H2 doublet, and sum (red) and separate ¹H1 (blue), ¹H2 (green) simulations. Simulation parameters (B/B model) are listed in Table 1. B) ¹H PESTRE traces collected from the ν_- (black) and ν₊ (purple) branch of the ¹H2 doublet. Experimental Conditions: A) ¹H stochastic CW ENDOR: same as in Fig. 4. ¹H Davies ENDOR: Microwave frequency, 34.757 GHz; π = 80 ns; τ = 600 ns; t_RF = 35 μs; repetition time, 50 ms; t_mix = 5 μs; temperature, 2 K. B) Same as in A (¹H Davies) except for: repetition time, 150 ms; RF frequency, 41.1 (ν_-) and 64.7 (ν₊) MHz.

Fig. 9.

2K 35 GHz^,2H Mims ENDOR spectra of H₂O and D₂O turnover samples at g₂ = 2.008. Experimental data (black and purple), and sum of simulations for^1,2H1 and^,2H2 (red). Mims holes in the ²H intensitiy indicated as open triangles show the extent of the ²H⁺ intensity. Simulation parameters for hydrides (B/B model) are listed in Table 1 and were scaled by factor g_N(²H)/g_N(¹H) = 6.5⁻¹ for ²H spectra. Experimental Conditions: A) Same as in Fig. 6. B) same as A except, microwave frequency, 34.80–34.87 GHz; repetition time, 100 ms; temperature.

Fig 10:

Reduced dipolar tensor components, a_i = T_i/t₁, calculated as a function of t₂/t₁ for a hydride bridge with R = 2.52Å, r₁ = 1.65,, r₂ = 1.69. The two a₁ nulls give similar angles, γ.

Chart 1:

¹H Tensor Permutations

Chart 2

Chart 3

Chart 4

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Chart 6