Resolving the structure of the E(1) 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 MoFe(7)S(9)C catalytic cluster to reduce N(2) to NH(3). This cluster requires the accumulation of three to four reducing equivalents prior to binding N(2); however, despite decades of research, the intermediate states formed prior to N(2) 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(1) 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(1) state, and use these metrics to assess a variety of possible models of the E(1) state. The combination of our experimental and theoretical results supports that formation of E(1) 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.