Resolving the structure of the E state of Mo nitrogenase through Mo and Fe K-edge EXAFS and QM/MM calculations.

Biological nitrogen fixation is predominately accomplished through Mo nitrogenase, which utilizes a complex MoFeSC catalytic cluster to reduce N to NH. This cluster requires the accumulation of three to four reducing equivalents prior to binding N; however, despite decades of research, the intermediate states formed prior to N binding are still poorly understood. Herein, we use Mo and Fe K-edge X-ray absorption spectroscopy and QM/MM calculations to investigate the nature of the E state, which is formed following the addition of the first reducing equivalent to Mo nitrogenase. By analyzing the extended X-ray absorption fine structure (EXAFS) region, we provide structural insight into the changes that occur in the metal clusters of the protein when forming the E state, and use these metrics to assess a variety of possible models of the E state. The combination of our experimental and theoretical results supports that formation of E involves an Fe-centered reduction combined with the protonation of a belt-sulfide of the cluster. Hence, these results provide critical experiment and computational insight into the mechanism of this important enzyme.