The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous L-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic C-13 hyperfine interactions for the two CN- ligands-a reporter of spin density on their respective coordinating iron ions collapses from approximate to 5.8 for the H-ox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [N-15]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.