Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

Abstract

Nitrogenase catalyzes the reduction of dinitrogen (N₂) to two ammonia (NH₃) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H₂. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H₂. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H₂ formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H₂O and D₂O, the proton inventory, was found to change linearly with the D₂O/H₂O ratio, revealing that a single H/D is involved in the rate-limiting step of H₂ formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H₂ formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H₂ formation where the S-H⁺ bond breaks and H⁺ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H₂ formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.

Figure 1

(A) Schematic representation of the FeMo-cofactor with α-Cys²⁷⁵, α-His⁴⁴² and R-homocitrate as ligands. The red highlighted square represents the catalytically active 4Fe-4S face of the FeMo-cofactor. (B) Shown are the E_n states of FeMo-cofactor during accumulation of the first four electrons/protons, along with the reductive elimination/oxidative addition (re/oa) mechanism at E₄(4H). The “2N2H” intermediate implies a species at the diazene reduction level of unknown structure and coordination geometry.

Figure 2

Cyclic voltammogram (CV) for wild type MoFe protein. CV for wild type MoFe protein was collected using CC as an electron mediator. Shown is the current density (j) as a function of the applied potential at different percentages of D₂O. The current was measured at −1.26 V, −1.25 V, −1.24, −1.24 V, and −1.24 V for 100% H₂O, 25% D₂O, 50% D₂O, 75% D₂O and 100% D₂O, respectively. The non-catalytic current was subtracted from the observed current to get the net catalytic current. In the inset, the ratio of net current at n fraction of D₂O (i_n) to the catalytic current in 0 % D₂O (i₀) is plotted against the mole fraction (n) of D₂O. The line is a fit of the data to the Gross-Butler equation for a one proton transfer (eqn 1), where the solvent isotope effect is 2.7. Condition: 250 mM HEPES pH/pD = 7.2), 667 μM CC and a scan rate of 2 mV/s at 23°C.

Figure 3

Cyclic voltammogram for wild type MoFe protein turnover (TO) under Ar (Ar TO) and turnover in nitrite (NO₂⁻ TO). Condition: 250 mM HEPES pH 7.2, 667 μM CC, 50 mM NO₂⁻ and scan rate of 2 mV/s at 23°C.

Figure 4

(Top) Kinetic scheme for accumulation of protons and electrons on FeMo-co and loss of H₂. The rate constants for ET are all taken to be k₁, while the rate constants for H₂ loss are k₂, k₃, and k₄. (Top) Shown is relaxation from E₄(4H) (solid line), E₃(3H) and E₂(2H); dashed lines indicate pathways suppressed in mediated electrochemistry (see text). (Bottom) Equivalent truncated catalytic cycle for H₂ formation involving E₀, E₁, and E₂.

Figure 5

Proton inventory plots (eq 6). (Left) Predicted plots of i_n/i₀ versus fraction of D₂O in buffer (n) at the indicated KIE_i for selected values of r; also, blue, 0.2; points, inventory as measured for [CC] = 50 μM (Right) A 3D plot of the proton inventory (eq 6) as function of (n, r).

Figure 6

Proton inventory plot MoFe proteins. Shown are the proton inventory plots for wild type (red), β-98^Tyr➔His (magenta), α-70^Val➔Ile (green), α-70^Val➔Ala/α-195^His➔Gln (blue) MoFe protein and nifX-FeMo-cofactor (black). Condition: 250 mM HEPES pH or pD 7.2, 667 μM CC, and scan rate of 2 mV/s at 23°C.

Figure 7

Structure of the E₂(2H) state and the transition state E₂(2H)^‡ for the release of H₂ (upper panels; for clarity, only the FeMo-co core is shown). Relevant distances are reported in Å along with the free energy of E₂(2H)^‡ and E₂(2D)^‡ relative to E₂(2H) and E₂(2D), respectively. The lower panels report the free energy ΔΔG of E₂(2D) and E₂(2D)^‡ relative to E₂(2H) and E₂(2H)^‡ along with the enthalpic (ΔΔH), entropic (−TΔΔS) and nuclear zero point energy (ΔΔZPE) contributions to ΔΔG. All energies in kJ/mol.