In Vivo Biogenesis of a De Novo Designed Iron-Sulfur Protein.

In vivo expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through in vitro reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron-sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters in vivo . While in vitro reconstitution produces a mixture of oligomers that depends on solution conditions, in vivo production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S] ²⁺ cluster. The multinuclear cluster of in vivo assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required in vivo, indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron-sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.