Significance [FeFe]-hydrogenases are H ₂ -forming enzymes with potential in renewable energy applications. Their molecular mechanism of catalysis needs to be understood. A protocol for specific ¹³ CO isotope editing of all carbon monoxide ligands at the six-iron cofactor (H-cluster) was established. Analysis of vibrational modes via quantum chemical calculations implies structural dynamics at the H-cluster in the active-ready state. Site-selective introduction of isotopic reporter groups opens new perspectives to identify intermediates in the catalytic cycle.