The active E4 structure of nitrogenase studied with different DFT functionals

The present study concerns the technical aspects of obtaining the energetics for the E4 state of nitrogenase, the enzyme that fixes N(2) in nature. EPR experiments have shown that the critical E4 structure that activates N(2) should contain two bridging hydrides in the FeMo‐cofactor. It is furthermore in equilibrium with a structure where the two hydrides have been released and N(2) binds. These observations led to the suggestion that E4 should have two bridging hydrides and two protonated sulfides. It is important to note that the structure for E4 has not been determined, but only suggested. For a long time, no DFT study led to the suggested structure, independent of which functional was used. However, in two recent DFT studies a good agreement with the experimental suggestion was claimed to have been obtained. In one of them the TPSS functional was used. That was the first out of 11 functionals tried that led to the experimentally suggested structure. In the second of the recent DFT studies, a similar conclusion was reached using the TPSSh functional. The conclusions in the recent studies have here been studied in detail, by calculating a critical energetic value strongly implied by the same EPR experiments. Both the TPSS and TPSSh functionals have been used. The present calculations suggest that those DFT functionals would not lead to agreement with the experimental EPR results either.