Protonation of Homocitrate and the E 1 State of Fe-Nitrogenase Studied by QM/MM Calculations

Nitrogenase is the only enzyme that can cleave the strong triple bond in N , making nitrogen available for biological life. There are three isozymes of nitrogenase, differing in the composition of the active site, viz., Mo, V, and Fe-nitrogenase. Recently, the first crystal structure of Fe-nitrogenase was presented. We have performed the first combined quantum mechanical and molecular mechanical (QM/MM) study of Fe-nitrogenase. We show with QM/MM and quantum-refinement calculations that the homocitrate ligand is most likely protonated on the alcohol oxygen in the resting E state. The most stable broken-symmetry (BS) states are the same as for Mo-nitrogenase, i.e., the three Noodleman BS7-type states (with a surplus of β spin on the eighth Fe ion), which maximize the number of nearby antiferromagnetically coupled Fe-Fe pairs. For the E state, we find that protonation of the S2B μ belt sulfide ion is most favorable, 14-117 kJ/mol more stable than structures with a Fe-bound hydride ion (the best has a hydride ion on the Fe2 ion) calculated with four different density-functional theory methods. This is similar to what was found for Mo-nitrogenase, but it does not explain the recent EPR observation that the E state of Fe-nitrogenase should contain a photolyzable hydride ion. For the E state, many BS states are close in energy, and the preferred BS state differs depending on the position of the extra proton and which density functional is used.