The Cyanide Ligands of [FeFe] Hydrogenase: Pulse EPR Studies of ~(13)C and ~(15)N-Labeled H-Cluster

摘要

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 ~(13)C hyperfine interactions for the two CN~-ligands-a reporter of spin density on their respective coordinating iron ions-collapses from ≈5.8 for the H_(ox) form of hydrogenase to ＜2 for the CO-inhibited form. Additionally, when the maturation was carried out using [~(15)N] -tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.