Asymmetry in the Ligand Coordination Sphere of the [FeFe] Hydrogenase Active Site Is Reflected in the Magnetic Spin Interactions of the Aza-propanedithiolate Ligand

[FeFe] hydrogenases are very active enzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. Their active site, the H-cluster, contains a unique binuclear iron complex, [2Fe]_H, with CN^- and CO ligands as well as an aza-propane-dithiolate (ADT) moiety featuring a central amine functionality that mediates proton transfer during catalysis. We present a pulsed ¹³C-ENDOR investigation of the H-cluster in which the two methylene carbons of ADT are isotope labeled with ¹³C. We observed that the corresponding two ¹³C hyperfine interactions are of opposite sign and corroborated this finding using density functional theory calculations. The spin polarization in the ADT ligand is shown to be linked to the asymmetric coordination of the distal iron site with its terminal CN^- and CO ligands. We propose that this asymmetry is relevant for the enzyme reactivity and is related to the (optimal) stabilization of the iron-hydride intermediate in the catalytic cycle.