Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the ⁵⁷Fe-based spectroscopic data—although rich with information—combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating ⁵⁷Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.