Chemical diversity of bilin species defines the chiroptical landscape of phytochromes

Phytochromes are widespread red/far-red light-sensing photoreceptors across several kingdoms of life that enable organisms to adapt their physiology to changing light environments. Major evolutionary transitions, from proteobacteria to cyanobacteria, algae and plants, imposed selective pressure on these proteins to diversify their spectral properties through changes in bilin cofactor preference. This adaptation coincided with a structural relocation of a cysteine residue, the covalent cofactor attachment site, from the N-terminal segment (NTS) to the GAF domain. Here, using the model protein Is PadC, we demonstrate how the shift from biliverdin- to phycocyanobilin-binding phytochromes modulates spectroscopic signatures while retaining overall functionality. We further resolve the long-standing question of divergent circular dichroism behavior between NTS _Cys and GAF _Cys phytochromes through a combination of wet-lab experiments and quantum chemical calculations. Contrary to a widely accepted model, we show that the geometric characteristics of the red-light-activated bilins follow a uniform mechanism across all classical phytochrome lineages. Spectral diversity, instead, arises from the chemical properties of the preferred bilin influenced by subtle structural deviations at the alternative attachment sites. These insights resolve a fundamental debate in phytochrome biology, demonstrate conserved functionality across different bilin species and provide a structural roadmap for color-tuning of phytochrome-based optogenetic tools.