Spectroscopic Evidence for Site Specific Chemistry at a Unique Iron Site of the [4Fe−4S] Cluster in Ferredoxin:Thioredoxin Reductase

Ferredoxin:thioredoxin reductase (FTR) catalyzes the reduction of the disulfide in thioredoxin in two one-electron steps using an active site comprising a [4Fe−4S] in close proximity to a redox active disulfide. Mössbauer spectroscopy has been used to investigate the ligation and electronic properties of the [4Fe−4S] cluster in as-prepared FTR which has the active-site disulfide intact and in the <i>N</i>-ethylmaleimide (NEM)-modified form which provides a stable analogue of the one-electron-reduced heterodisulfide intermediate and has one of the cysteines of the active-site disulfide alkylated with NEM. The results reveal novel site-specific cluster chemistry involving weak interaction of the active-site disulfide with a unique Fe site of the [4Fe−4S]²⁺ cluster in the resting enzyme and cleavage of the active-site disulfide with concomitant coordination of one of the cysteines to yield a [4Fe−4S]³⁺ cluster with a five-coordinate Fe site ligated by two cysteine residues in the NEM-modified enzyme. The results provide molecular-level insight into the catalytic mechanism of FTR and other Fe−S-cluster-containing disulfide reductases, and suggest a possible mechanism for the reductive cleavage of S-adenosylmethionine by the radical SAM family of Fe−S enzymes.