Photoinduced Reductive Elimination of H2 from the Nitrogenase Dihydride (Janus) State Involves a FeMo-cofactor-H2 Intermediate

Abstract

N₂ reduction by nitrogenase involves the accumulation of four reducing equivalents at the active site FeMo-cofactor to form a state with two [Fe-H-Fe] bridging hydrides (denoted E₄(4H), the Janus intermediate), and we recently demonstrated that the enzyme is activated to cleave the N≡N triple bond by the reductive elimination (re) of H₂ from this state. We are exploring a photochemical approach to obtaining atomic-level details of the re activation process. We have shown that, when E₄(4H) at cryogenic temperatures is subjected to 450 nm irradiation in an EPR cavity, it cleanly undergoes photoinduced re of H₂ to give a reactive doubly reduced intermediate, denoted E₄(2H)*, which corresponds to the intermediate that would form if thermal dissociative re loss of H₂ preceded N₂ binding. Experiments reported here establish that photoinduced re primarily occurs in two steps. Photolysis of E₄(4H) generates an intermediate state that undergoes subsequent photoinduced conversion to [E₄(2H)* + H₂]. The experiments, supported by DFT calculations, indicate that the trapped intermediate is an H₂ complex on the ground adiabatic potential energy suface that connects E₄(4H) with [E₄(2H)* + H₂]. We suggest that this complex, denoted E₄(H₂; 2H), is a thermally populated intermediate in the catalytically central re of H₂ by E₄(4H) and that N₂ reacts with this complex to complete the activated conversion of [E₄(4H) + N₂] into [E₄(2N2H) + H₂].

Figure 1

Representations of the mechanistically central re/oa equilibrium that activates nitrogenase to break the N≡N triple bond (upper), and of the photoinduced re/oa equilibrium (lower). These cartoons represent the Fe 2,3,6,7 face of FeMo-co; but we emphasize they are not meant to be ‘anatomically’ precise.

Figure 2

Comparison of normalized 12 K and 50 K time courses of E₄(4H) (red) and E₄(2H)* (black) states during irradiation of α-70^Val→Ile H₂O turnover sample with 450 nm laser diode light. Solid lines are to guide the eye; dotted lines show the lag in E₄(2H)* formation at 12 K.

Figure 3

Results of 77 K annealing of α-70^Val→Ile turnover irradiated at various temperatures. (*)-labeled features of EPR spectra indicate recovery of dihydride intermediate state E₄(4H) upon annealing (left) in parallel with disappearance of photoinduced species (right).

Figure 4

Subtraction-elimination of E₄(4H) and E₄(2H)* signals from EPR spectrum of α-70^Val→Ile turnover irradiated at 12 K (red) with use of combination of spectra recorded before irradiation (black) and after 77 K annealing, which followed the irradiation (green). Result presents otherwise unresolved new photoinduced signal B (blue) unstable at 77K.

Fig 5. Alternative 2-step mechanisms for photoinduced reaction

(Top) re; assignment of intermediate discussed below; (middle, bottom) alternative mechanisms, see text.

Figure 6

Idealized energy landscape for the low-temperature photoinduced re/oa of the Janus intermediate, E₄(4H). Excited-state surface: the proposed identifications of the states A†, B, and B† are discussed in the text. Yellow arrows, photoexcitation; black arrows, relaxation, red arrow, nuclear tunneling.

Figure 7

Normalized time courses of E₄(4H) (red) and E₄(2H)* (black) states measured during photolysis at 12 K (upper) and 50 K (lower) are shown fitted as previously described in accordance with corresponding low and high temperature kinetic schemes. Data points for photoinduced intermediate B at 12 K were obtained with subtraction procedure described in Figs 4, S1, and are shown in comparison with the kinetic scheme prediction (blue trace). Low accumulation of B state at 50 K is due to back reaction step with time constant τ₃ = 160 sec.

Figure 8

Antibonding excited state of bridging hydride.

Figure 9

Cartoon version of suggested catalytic pathway for re/oa activation of FeMo-co for N₂ reduction.

Scheme 1

Scheme 2