What Is the Structure of the E4 Intermediate in Nitrogenase?

Lili Cao,Ulf Ryde

doi:10.1021/acs.jctc.9b01254

Abstract

Nitrogenase is the only enzyme that can cleave the strong triple bond in N2. The active site contains a complicated MoFe7S9C cluster. It is believed that it needs to accept four protons and electrons, forming the E4 state, before it can bind N2. However, there is no consensus on the atomic structure of the E4 state. Experimental studies indicate that it should contain two hydride ions bridging two pairs of Fe ions, and it has been suggested that both hydride ions as well as the two protons bind on the same face of the cluster. On the other hand, density functional theory (DFT) studies have indicated that it is energetically more favorable with either three hydride ions or with a triply protonated carbide ion, depending on the DFT functional. We have performed a systematic combined quantum mechanical and molecular mechanical (QM/MM) study of possible E4 states with two bridging hydride ions. Our calculations suggest that the most favorable structure has hydride ions bridging the Fe2/6 and Fe3/7 ion pairs. In fact, such structures are 14 kJ/mol more stable than structures with three hydride ions, showing that pure DFT functionals give energetically most favorable structures in agreement with experiments. An important reason for this finding is that we have identified a new type of broken-symmetry state that involves only two Fe ions with minority spin, in contrast to the previously studied states with three Fe ions with minority spin. The energetically best structures have the two hydride ions on different faces of the FeMo cluster, whereas better agreement with ENDOR data is obtained if they are on the same face; such structures are only 6–22 kJ/mol less stable.

Highlights

Nitrogenase (EC 1.18/19.6.1) is the only enzyme that can cleave the triple bond of N2, forming two molecules of ammonia and making atmospheric nitrogen available for biological systems.[1−3] Nitrogenase is found only in some bacteria and cyanobacteria, but several higher plants, e.g. legumes, rice, and alder, live in symbiosis with such microorganisms
The electronic structure of all QM calculations was obtained with the broken-symmetry (BS) approach:[20] Each of the seven Fe ions were modeled in the high-spin state, with either a surplus of α or β spin
We investigate whether it is possible to find a structure of the E4 state in nitrogenase that is consistent with the experimental ENDOR data,[3,29] i.e., with two bridging hydride ions, and that is lower in energy than other possible structures

Summary

■ INTRODUCTION

Nitrogenase (EC 1.18/19.6.1) is the only enzyme that can cleave the triple bond of N2, forming two molecules of ammonia and making atmospheric nitrogen available for biological systems.[1−3] Nitrogenase is found only in some bacteria and cyanobacteria, but several higher plants, e.g. legumes, rice, and alder, live in symbiosis with such microorganisms. We perform a more systematic search for the best E4 structure with two bridging hydride ions and two protonated sulfides, using combined QM/MM calculations[36] and methods to deal with the broken-symmetry states and other computational difficulties developed in our previous studies.[33,34,37]. The electronic structure of all QM calculations was obtained with the broken-symmetry (BS) approach:[20] Each of the seven Fe ions were modeled in the high-spin state, with either a surplus of α (four Fe ions) or β (three Fe ions) spin. A starting wave function for the FeMo cluster was obtained by first optimizing the all-high-spin state with 35 unpaired electrons and changing the total α and β occupation numbers to the desired net spin. The geometry optimizations were continued until the energy change between two iterations was less than 2.6 J/mol (10−6 a.u.), and the maximum norm of the Cartesian gradients was below 10−3 a.u

■ RESULT AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

Findings

■ REFERENCES