H 2 formation from the E 2 -E 4 states of nitrogenase.

Nitrogenase is the only enzyme that can cleave the strong triple bond in N ₂ , making nitrogen available for biological lifeforms. The active site is a MoFe ₇ S ₉ C cluster (the FeMo cluster) that binds eight electrons and protons during one catalytic cycle, giving rise to eight intermediate states E ₀ -E ₇ . It is experimentally known that N ₂ binds to the E ₄ state and that H ₂ is a compulsory byproduct of the reaction. However, formation of H ₂ is also an unproductive side reaction that should be avoided, especially in the early steps of the reaction mechanism (E ₂ and E ₃ ). Here, we study the formation of H ₂ for various structural interpretations of the E ₂ -E ₄ 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 H ₂ cannot form from such structures. On the other hand, with TPSS, r ² SCAN and TPSSh, H ₂ formation is strongly exothermic for all structures and E _n and therefore need strict kinetic control to be avoided. For the E ₂ state, the kinetic barriers for the low-energy structures are high enough to avoid H ₂ formation. However, for both the E ₃ and E ₄ 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 H ₂ with an activation barrier of only 29-57 kJ mol ^-1 , corresponding to rates of 7 × 10 ² to 5 × 10 ⁷ 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 ).