H2 formation from the E2–E4 states of nitrogenase

Abstract

Nitrogenase is the only enzyme that can cleave the strong triple bond in N₂, making nitrogen available for biological lifeforms. The active site is a MoFe₇S₉C cluster (the FeMo cluster) that binds eight electrons and protons during one catalytic cycle, giving rise to eight intermediate states E₀–E₇. It is experimentally known that N₂ binds to the E₄ state and that H₂ is a compulsory byproduct of the reaction. However, formation of H₂ is also an unproductive side reaction that should be avoided, especially in the early steps of the reaction mechanism (E₂ and E₃). Here, we study the formation of H₂ for various structural interpretations of the E₂–E₄ states using combined quantum mechanical and molecular mechanical (QM/MM) calculations and four different density-functional theory methods. We find large differences in the predictions of the different methods. B3LYP strongly favours protonation of the central carbide ion and H₂ cannot form from such structures. On the other hand, with TPSS, r²SCAN and TPSSh, H₂ formation is strongly exothermic for all structures and E_n and therefore need strict kinetic control to be avoided. For the E₂ state, the kinetic barriers for the low-energy structures are high enough to avoid H₂ formation. However, for both the E₃ and E₄ states, all three methods predict that the best structure has two hydride ions bridging the same pair of Fe ions (Fe2 and Fe6) and these two ions can combine to form H₂ with an activation barrier of only 29–57 kJ mol⁻¹, corresponding to rates of 7 × 10² to 5 × 10⁷ s⁻¹, i.e. much faster than the turnover rate of the enzyme (1–5 s⁻¹). We have also studied H-atom movements within the FeMo cluster, showing that the various protonation states can quite freely be interconverted (activation barriers of 12–69 kJ mol⁻¹).