Designed Rubredoxin miniature in a fully artificial electron chain triggered by visible light

Designing metal sites into de novo proteins has significantly improved, recently. However, identifying the minimal coordination spheres, able to encompass the necessary information for metal binding and activity, still represents a big challenge, today. Here, we tested our understanding with a benchmark, nevertheless difficult, case. We assembled in a small 28-residue peptide the quintessential elements required to correctly fold around a single iron redox center, coordinated to four cysteinyl thiolates (FeCys4 site), and to efficiently function in electron-transfer. This study represents a milestone in de novo protein design: for the first time the crystal structure of a designed tetra-thiolate metal-binding protein is reported within sub-Å agreement with the intended design. This allowed us to well correlate structure to spectroscopic and electrochemical properties. Given its high reduction potential compared to natural and designed FeCys4-containing proteins, we exploited it as terminal electron acceptor of a fully artificial chain triggered by visible light.