Climbing nitrogenase: toward a mechanism of enzymatic nitrogen fixation

Abstract

"Nitrogen fixation", the reduction of dinitrogen (N2) to two ammonia (NH3) molecules, by the Mo-dependent nitrogenase is essential for all life. Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase activity make it the "Everest of enzymes". This Account describes our efforts to climb one "face" of this mountain by meeting two interdependent challenges central to determining the mechanism of biological N2 reduction. The first challenge is to determine the reaction pathway: the composition and structure of each of the substrate-derived moieties bound to the catalytic FeMo cofactor (FeMo-co) of the molybdenum-iron (MoFe) protein of nitrogenase. To overcome this challenge, it is necessary to discriminate between the two classes of potential reaction pathways: (1) a "distal" (D) pathway, in which H atoms add sequentially at a single N or (2) an "alternating" (A) pathway, in which H atoms add alternately to the two N atoms of N2. Second, it is necessary to characterize the dynamics of conversion among intermediates within the accepted Lowe-Thorneley kinetic scheme for N2 reduction. That goal requires an experimental determination of the number of electrons and protons delivered to the MoFe protein as well as their "inventory", a partition into those residing on each of the reaction components and released as H2 or NH3. The principal obstacle to this "climb" has been the inability to generate N2 reduction intermediates for characterization. A combination of genetic, biochemical, and spectroscopic approaches recently overcame this obstacle. These experiments identified one of the four-iron Fe-S faces of the active-site FeMo-co as the specific site of reactivity, indicated that the side chain of residue alpha70V controls access to this face, and supported the involvement of the side chain of residue alpha195H in proton delivery. We can now freeze-quench trap N2 reduction pathway intermediates and use electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize them. However, even successful trapping of a N2 reduction intermediate occurs without synchronous electron delivery to the MoFe protein. As a result, the number of electrons and protons, n, delivered to MoFe during its formation is unknown. To determine n and the electron inventory, we initially employed ENDOR spectroscopy to analyze the substrate moiety bound to the FeMo-co and 57Fe within the cofactor. Difficulties in using that approach led us to devise a robust kinetic protocol for determining n of a trapped intermediate. This Account describes strategies that we have formulated to bring this "face" of the nitrogenase mechanism into view and afford approaches to its climb. Although the summit remains distant, we look forward to continued progress in the ascent.

Fig. 1

Early stages of Lowe-Thorneley (LT) kinetic scheme for N₂ reduction, the portion that connects states E₀ through E₄; for a full scheme, see references –. As discussed in conjunction with Fig 8, A is the MoFe protein 'hydride' intermediate; B forms during its relaxation to resting state, C.

Fig 2

Structure of FeMo-co, including the two residues that covalently link it to the apo-protein and the two implicated in function, α-V70 as substrate 'gatekeeper' and α-H195 as agent for proton delivery.

Fig 3

Proposed structure for the intermediate trapped during propargyl alcohol reduction – the product alkene bound side on to a single Fe ion.

Fig 4

‘Alternating’ (A) and 'Distal' (D) N₂ reduction pathways. M represents FeMo-co without specifying metal ion(s) involved. Likewise, the representations of binding are meant to emphasize the distinction between pathways and do not imply specific binding modes. Small straight arrows represent addition of H⁺/e⁻ to substrate.

Fig 5

(Left) Q-band Mims (e and m) and Re-Mims (l) ¹⁵N-ENDOR spectra (g₁) of nitrogenous trapped intermediates, labeled with ¹⁵N as indicated. Conditions: microwave frequency = 34.808–34.819 GHz; π/2 = 52 ns (e, m) and 32 ns (l); RF = 20 ~ 30 µs; τ = 500 ns (e), 300 ns (m), and 200 ns (l); sampling = ~ 1000 transients/point; repetition rate = 100 Hz (e and m), 50 Hz (l); 2 K. (Right) CW ¹H-ENDOR of ¹⁴N intermediates in H₂O and D₂O. Conditions: microwave frequency, 35.057–35.171 GHz; modulation amplitude = 4 G; RF sweep speed = 1 MHz/s; bandwidth of RF broadened to 100 kHz; 2K.

Fig 6

Cartoon representation of alternate schemes for binding C₂H₄ to a cofactor metal ion. Left: dative π bonding. Right: oxidative addition to form Fe-C σ bonds.

Fig 7

3D representation of LT scheme plotted in terms of the proton inventory (eq 4), showing alternate limiting reaction pathways for N₂ reduction by nitrogenase following N₂ binding to intermediate E₄ of Fig 1. Indices of intermediate states are: n (abscissa); h_s (ordinate) = number of protons delivered to substrate; r (z axis) = number of electrons/protons release during catalysis.

Fig 8

Step-annealing curves (253K) of hydride intermediate (A), leading to formation of B intermediate and ground state C, in H₂O/D₂O buffers. Fits to distributed (stretched exponential) two-step relaxation: t₁(H₂O) = 13 min, t₁(D₂O) = 49 min; t₂(H₂O) = 870 min, t₂(D₂O) = 2700 min. For clarity, data for A(H) and A(D) are omitted, and only the fits are presented.

Chart 1

Substrate-derived species bound to FeMo-co that might form in late stages of N₂ reduction.