Mechanisms of Red-Shifted Absorption in Biliverdin-Binding Proteins.

Biliverdin (BV) is a linear tetrapyrrole chromophore that enables far-red and near-infrared photoreception in phytochromes and fluorescence in engineered probes. Here, we systematically investigate a broad set of BV-binding proteins using a consistent quantum mechanical/molecular mechanical framework and demonstrate that their absorption wavelengths can be quantitatively reproduced across both BV- and phycocyanobilin (PCB)-binding proteins. In contrast to PCB-binding proteins, BV-binding proteins show a substantially weaker dependence of absorption wavelength on chromophore coplanarity. Instead, the BV color tuning arises from the combined effects of chromophore conformation, electrostatic interactions, desolvation, and π-stacking. Inverted D-ring geometries in Agp2-PCM and Agp2-PAiRFP2 are associated with pronounced red shifts; tryptophan-mediated π-stacking contributes to additional red shifts in JSC1_58120g3-type proteins, and, in sandercyanin, high solvent exposure leads to substantially smaller desolvation-induced blue shifts. Collectively, in contrast to PCB-binding proteins, where absorption trends are largely governed by chromophore shape, BV-binding proteins show relatively stronger contributions from additional factors such as electrostatic interactions and desolvation.