Reversible Photoinduced Reductive Elimination of H2 from the Nitrogenase Dihydride State, the E(4)(4H) Janus Intermediate

Abstract

We recently demonstrated that N2 reduction by nitrogenase involves the obligatory release of one H2 per N2 reduced. These studies focus on the E4(4H) "Janus intermediate", which has accumulated four reducing equivalents as two [Fe-H-Fe] bridging hydrides. E4(4H) is poised to bind and reduce N2 through reductive elimination (re) of the two hydrides as H2, coupled to the binding/reduction of N2. To obtain atomic-level details of the re activation process, we carried out in situ 450 nm photolysis of E4(4H) in an EPR cavity at temperatures below 20 K. ENDOR and EPR measurements show that photolysis generates a new FeMo-co state, denoted E4(2H)*, through the photoinduced re of the two bridging hydrides of E4(4H) as H2. During cryoannealing at temperatures above 175 K, E4(2H)* reverts to E4(4H) through the oxidative addition (oa) of the H2. The photolysis quantum yield is temperature invariant at liquid helium temperatures and shows a rather large kinetic isotope effect, KIE = 10. These observations imply that photoinduced release of H2 involves a barrier to the combination of the two nascent H atoms, in contrast to a barrierless process for monometallic inorganic complexes, and further suggest that H2 formation involves nuclear tunneling through that barrier. The oa recombination of E4(2H)* with the liberated H2 offers compelling evidence for the Janus intermediate as the point at which H2 is necessarily lost during N2 reduction; this mechanistically coupled loss must be gated by N2 addition that drives the re/oa equilibrium toward reductive elimination of H2 with N2 binding/reduction.

Figure 1

Crystal structure of FeMo-co. Fe is show in rust, Mo in magenta, S in yellow, carbide in dark-grey, carbon in gray, N in blue and O in red. The Fe atoms of catalytic 4Fe-4S face are labelled as 2, 3, 6, and 7. Two amino acids, α-70^Val and α-195^His, around the FeMo-co are also shown; the former or both are modified in enzyme used in this study (see text). The image was created using PDB coordinate 2AFI.

Figure 2. Schematic of re/oa Equilibrium

The cartoon represents the Fe 2,3,6,7 face of FeMo-co, and the ‘2N2H’ implies a species at the diazene reduction level of unknown structure and coordination geometry. In the indicated equilibrium the binding and activation of N₂ is mechanistically coupled to the re of H₂, as described in the text. In the E_n notation, n = number of [e⁻/H⁺] added to FeMo-co; parenthesis denotes the stoichiometry of H/N bound to FeMo-co.

Figure 3

Schematic of alternative limiting mechanisms for re/oa equilibrium.

Figure 4

X-band EPR spectra of MoFe protein (α-70^Ile) freeze-trapped during Ar turnover in H₂O before (black) and during irradiation with 450 nm diode laser at 12 K (blue (2.5 min) and red (20 min) traces); Red arrows highlight the conversion of E₄(4H) to the photoinduced S state. Dashed Green spectrum shows that annealing (Ann) the illuminated sample at 217 K for 2 minutes causes complete reversion of S to E₄(4H). EPR conditions: T = 12 K; microwave frequency, 9.36 GHz; microwave power, 10 mW; modulation amplitude, 13 G; time constant, 160 ms; field sweep speed, 20 G/s.

Figure 5

Q-band stochastic ¹H CW ENDOR spectra showing loss of signals from hydrides, H1 and H2, through photolysis. (Black) Before and (red) after 450 nm photolysis of MoFe protein (α-70^Ile/α-195^Gln) trapped during Ar turnover in H₂O buffer. ENDOR conditions: microwave frequency, ~34.99 GHz; modulation amplitude, 6.3 G; RF duration 3 ms; RF cycle, 200 Hz; bandwidth of RF broadened to 100 kHz; 2000 scans; temperature, 2 K.

Figure 6

Alternative mechanisms for the E₄(4H) ⇒ S photo-conversion through loss of both hydrides and release of H₂ and thermal reverse.

Figure 7

Decay during 193 K annealing of E₄(2H)* photoinduced in MoFe (α-70^Ile) freeze-trapped during turnover in H₂O (red) and D₂O (blue), along with the parallel recoveries of E₄(4H). Data points obtained as intensities of g₁ feature of the corresponding S=1/2 EPR signals, normalized to the maximum signal; they were fit with an exponential function, with time constants shown in the figure. EPR conditions: as in Figure 4.

Figure 8

Timecourse of in situ 450 nm photoinduced conversion of E₄(4H) intermediate trapped during MoFe protein (α-70^Ile) turnover in H₂O (lower) and D₂O (upper). Photolysis at 3.8 K (green), 8 K (red) and 12 K (blue). Signal measured directly as intensity of the g₁ feature of the E₄(4H) S=1/2 EPR signal, normalized to the maximum signal and fit with a stretched exponential decay function, I = exp(−[t/τ]ⁿ), with ‘1/e’ time constant, τ; 0 < n ≤ 1 equals unity for exponential decay and decreases with the spread of the distribution. Time constants for fits (white dashed lines) are given in figure; in all cases n ~ 0.4 (see SI for details). EPR conditions: microwave frequency, 9.36 GHz; microwave power, 10 mW (1 mW for measurements at 3.8 K); modulation amplitude, 13 G; time constant, 160 ms.

Figure 9

Idealized energy surfaces for photoinduced re/oa of the Janus intermediate, E₄(4H). Among issues to be resolved are the possibility of tunneling on the excited-state surface, ‘(?)’, and whether there are stable intermediates along re or oa paths.

Figure 10

Cartoon showing nodal properties of an excited MO for an M(H)₂ complex that is bonding between the two hydrides and antibonding between each one and the metal dz² orbital.

Figure 11

Cartoon showing nodal properties of the excited MO’s for bridging hydrides (parallel arrangement), each antibonding between the hydride and the two dz² orbitals on the Fe ions it bridges.

Chart 1