Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N₂ for H₂

Abstract

Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During -50 °C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e(-)/H(+)] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3.

Figure 1

(A) Simplified LT kinetic scheme highlighting correlated electron/proton delivery in eight steps, with inclusion of some of the possible pathways for decay by H₂ release; although N₂ binds at either the E₃ or E₄ levels, only the E₄ state is reactive, so the pathway through the E₄ state is emphasized. For states under discussion in this report, parentheses added to the E_n notation to denote the stoichiometry of H/N bound to FeMo-co. When the molecular species corresponding to this stoichiometry is specified, it is noted; for example, E₄(2N2H) might have N₂ or N₂H₂ bound, and would be notated as E₄(N₂,2H) and E₄(N₂H₂). (B) The reductive-elimination (re) mechanism for H₂ release upon N₂ (blue) binding to E₄(4H) and its reverse, oxidative-addition (oa) of H₂ with loss of N₂ (eq 4) visualized as occurring on the FE2,3,6,7 face of FeMo-co. The binding modes of the hydrides of E₄(4H) and the components of diazene in E₄(2N2H) are arbitrary.

Figure 2

Selected EPR spectra collected during the time course for −50°C cryoannealing of a sample freeze-trapped under P(N₂) = 0.05 atm, and showing the decompositions of the S = 3/2 spectra in the low-field g₁g₂ region into contributions from 1a (g = [4.32, 3.66, 2.01], red) and 1b (g = [4.21, 3.76, ~1.97], blue). At early time the g-2 region is dominated by EG, as indicated. The indicated signal from the reduced 4Fe/4S cluster of Fe protein is greatly distorted at this temperature, but it is clear that its intensity does not change with annealing. EPR conditions: temperature, 3.8 K; microwave frequency, 9.36 GHz; microwave power, 0.5 mW; modulation amplitude, 13 G; time constant, 160 ms; field sweep speed, 38 G/s.

Figure 3

(A) Decay of intermediate EG freeze-trapped during turnover of nitrogenase under P(N₂) = 0.05 (open circles; see Figure 2) and 1 atm (solid circles) observed during cryoannealing at −50 °C. Plotted are intensities of g₁ feature of the S = ½ EPR signal, shown after normalization to the maximum (zero-time) signal and fitted with a stretched exponential decay function, eq 2. EPR conditions: as for Figure 2, except for field sweep speed of 20 G/s. (B) Progress curves for the three EPR-active species, EG, 1a, and 1b, observed during cryoannealing of EG freeze-trapped under P(N₂) = 0.05 atm. Fits to kinetic scheme (eq 3) as previously described; parameters of k₁ for EG stretched-exponential decay presented in 3A; rate of the second step of eq 3, k₂ = 0.0023 min⁻¹.^, Intensities for the resting state (black) and E₂ state (blue) obtained with the previously described procedure of deconvolution and quantitation of corresponding S = 3/2 EPR signals 1a and 1b (see Figure 2). Intensities for the EG intermediate (red) taken from Figure 3A and converted to concentration units by scaling to the two-step kinetic scheme for decay, eq 3. EPR conditions: as for Figure 3A, except for field sweep speed of 38 G/s for 1a and 1b signals detection.

Figure 4

Progress curves for −50 °C cryoannealing of EG intermediates formed during turnover under P(N₂) = 0.1 (open circles) and 1 (closed circles) atm, with (red) or without (black) stirring reaction mixture. Blue arrows connect pairs of samples prepared with different P(N₂), with arrow pointing towards higher solution [N₂], namely P(N₂) = 1 atm; within a pair, [H₂] is comparable, either low because in both samples enzymatically produced H₂ has been flushed out by stirring, or high in unstirred samples; green arrows connect pairs of samples prepared under the same P(N₂), and thus with comparable [N₂] (either low because P(N₂) = 0.1 atm or high, P(N₂) = 1 atm) but different [H₂], with arrow pointing from sample in which [H₂] is low because stirring has flushed out the H₂ into the headspace, towards unstirred sample with higher [H₂]. Intensities measured as described in Figure 3A. Decays are fit as stretched exponentials (eq 2) with the parameters in the following table: [Table: see text]

Figure 5

Variation in the EPR amplitude of trapped intermediate EG g₁ feature at various N₂ partial pressures when reaction mixture is neither stirred nor flushed (red); stirred (green); stirred with the headspace of reaction vial flushed with appropriate N₂/Ar mixture during turnover. EPR conditions: as described in Figure 3A.

Scheme 1

Kinetic scheme for the decay of freeze-trapped E₄(2N2H) derived from Figure 1A; k_r and k_b are the second-order rate constants for re and its reverse; k_d and k_d’ are the rate constants for the irreversible decay of E₄(4H) and E₂(2H), respectively.