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The radical SAM enzyme superfamily of more than 50,000 enzymes, is recognized for its signature CxxxCxxC motif (C = cysteine, x = amino acid), that binds an iron sulfur cluster and cleaves S-adenosyl-L-methione (SAM), resulting in 5-deoxyadenosyl radical. The reactivity of these important catalysts has begun to be characterized by pulsed electron paramagnetic resonance (EPR) spectroscopy. The advanced methods of EPR that manipulate electron and nuclei spin interactions are a key means to understanding the one electron chemistry of radical SAM enzymes. The presented thesis work on biotin synthase is representative of the applications of EPR in the study of the radical SAM superfamily. Biotin synthase is a radical SAM enzyme that has been extensively studied on but its story is still incomplete. With pulsed EPR, the most recent research supports the proposal that the intermediate, 9-mercaptodethiobiotin (9-MDTB) obtains a sulfur from the nearby [2Fe-2S]2+ cluster when it is ligated to dethiobiotin (DTB). The core of the work presented here focuses on determining the isotropic and anisotropic hyperfine couplings of the cysteine beta-protons of the [2Fe-2S]2+ cluster of the intermediate 9-MDTB. The intermediate is a huge step in understanding radical SAM enzymes. The electronic structure was explored via isotopically labeled 15N 9-MDTB with pulsed EPR methods ENDOR and HYSCORE. Using multiple pulsed methods is useful to support the information gathered from each method. Both ENDOR and HYSCORE spectra were collected to derive the possible spin density of the Fe(III) and Fe(II) ions of the reduced 2Fe-2S]+ cluster as well as the possible dihedral angles of the ligand environment of the [2Fe-2S]+ cluster. The hyperfine couplings, spin densities, and dihedral angles are consistent with previously studied [2Fe-2S]+ clusters.