Design of a miniature protein in a fully artificial electron transport chain triggered by visible light

The design of de novo metal-binding proteins tailored for specific applications is continuously improving. The identification of the least set of primary and secondary sphere interactions around the metal able to delineate the observed function still challenges the bioinorganic chemistry community. Here, we benchmarked our understanding with a very well-studied, but at the same time insidious, case. We report the design of a single-chain linear protein able to recover all the requirements needed to fold and function as a natural Rubredoxin (FeCys4 site) in only 28 residues. Despite the simplicity of this metal site, de novo proteins featuring tetrathiolate metal clusters have never been characterized before by single-crystal X-ray diffraction. In this study, we show, for the first time in de novo protein design, the crystal structure of a tetra-thiolate metal-binding protein within sub-Å agreement with the intended design. Despite sharing strikingly similar structural arrangement, this miniaturized protein does not hold any sequence correlation to the known Rds. As a further achievement, we purposely programmed a high reduction potential compared to natural and designed FeCys4-containing proteins, and we exploited it as terminal electron acceptor of a fully artificial electron transport chain triggered by visible light.