Reductive Elimination of H₂ Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E₄(4H) Janus Intermediate in Wild-Type Enzyme

Abstract

We have proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70Val→Ile substituted MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN=NH) reduction level bound to the catalytic FeMo-cofactor. In the present work, EPR/ENDOR and photophysical measurements show that a state freeze-trapped during N2 reduction by wild type (WT) MoFe protein is the same Janus intermediate, thereby establishing the α-70Val→Ile intermediate as a reliable guide to mechanism, and enabling new experimental tests of the re/oa mechanism with WT enzyme. These allow us to show that the re/oa mechanism accounts for the longstanding Key Constraints on mechanism. Monitoring the S = ½ FeMo-co EPR signal of Janus in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby explaining earlier observations and verifying the re/oa mechanism. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. The relative populations of E4(2N2H) and E4(4H) freeze-trapped during WT turnover furthermore show that the rapidly reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is roughly thermoneutral (ΔreG0 ~ −2 kcal/mol), whereas hydrogenation of gas-phase N2 would be highly endergonic. These findings establish (i) that re/oa satisfies all key constraints on mechanism, (ii) that Janus is the key to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus emerges a picture of the central mechanistic steps by which the nitrogenase MoFe protein carries out one of the most challenging chemical transformation in biology, the reduction of the N≡N triple bond.

Hydride Conformers of the Nitrogenase FeMo-cofactor Two-Electron Reduced State E2(2H), Assigned Using Cryogenic Intra Electron Paramagnetic Resonance Cavity Photolysis

“…This suggests that the two hydride conformers are able to readily interconvert during fluid-solution turnover. In contrast, in freeze-trapped samples, we have only detected one form of the key E 4 (4H) Janus intermediate, 4,6 suggesting that this single conformer is “tuned” to carry out re of the hydrides and the subsequent binding/reduction of N 2 with a loss of H 2 . 4,9 …”

“…Our earlier conclusion that 1b is the E 2 (2H) state with its reducing equivalents stored as a bound hydride 4,6,9 is corroborated by the observation that the E 2 /1b state is photoactive, like inorganic hydride complexes, 17,18 the [Fe–H–Fe] bridging hydrides of the E 4 (4H) state of nitrogenase, 19,20 and the [Ni–H–Fe] bridging hydrides of the Ni–C state of NiFe hydrogenase. 21 As noted above, the earlier cryoannealing study of E 4 (4H) implied that the hydride of E 2 /1b adopts a [Fe–H–Fe] bridging structure, and it will be thus described in this report.…”

“…Conversely, it confirms that the FeMo-co metal ion core cannot be doubly reduced except at the E 4 state through the re of H 2 . 4,9 …”

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

“…Our initial report showed that E 4 (4H) exhibits the photoinduced re/oa equilibrium of Fig 1, 12 and we subsequently showed that the E 4 (4H) in the α-70 Val→Ile variant and the WT enzyme are identical in properties and behavior. 4 …”

“…4 The protein was obtained from the corresponding Azotobacter vinelandii strains as described elsewhere. 24 The handling of all buffers and proteins were done anaerobically under Ar atmosphere or under Schlenk vacuum line unless stated otherwise.…”