Functional Coordination of Iron-Sulfur Clusters by Selenocysteine

Iron-sulfur clusters are widely recruited by proteins throughout nature, playing a critical role in oxidative phosphorylation. Canonically, iron-sulfur clusters are coordinated by cysteine residues and (rarely) by histidine residues. Here we describe replacement of four cysteine residues required for iron-sulfur cluster coordination with selenocysteine (Sec), the 21^st amino acid. This substitution was accomplished using an engineered tRNA designed for EF-Tu mediated Sec insertion through stop codon suppression.

In E. coli protein Formate Dehydrogenase H (FDH_H), residues Cys8, Cys11, Cys15, and Cys42 coordinate a [4Fe-4S] cluster essential for protein function. We show that single substitution all four residues, and simultaneous substitution of up to three residues, results in production of functional protein in vivo. Kinetic assays of proteins with a single residue replaced show no significant change in activity.