Roles of individual pigments in ultrafast excitation dynamics of light-harvesting phycobiliproteins revealed by recombinant techniques and two-dimensional electronic spectroscopy

Phycobiliproteins serve as highly efficient light-harvesting antennae in cyanobacteria, yet the molecular factors governing their ultrafast energy relaxation and coherence dynamics remain incompletely understood. In this study, we investigate the role of pigment arrangement and pigment-protein interactions by combining recombinant protein engineering with two-dimensional electronic spectroscopy (2D-ES). In addition to wild-type allophycocyanin (APC) and C-phycocyanin (CPC), we artificially synthesized a {\beta}153 phycocyanobilin (PCB)-deficient CPC mutant, enabling direct experimental isolation of the contribution of this peripheral pigment. The absorption and fluorescence spectra show that removal of the {\beta}153 pigment primarily eliminates its spectral contribution without significantly altering the excitonic coupling between the {\alpha}84 and {\beta}84 pigments. Time-resolved 2D-ES reveals the close similarity between the dynamics of wild-type and {\beta}153-deficient CPCs, which demonstrate that the {\beta}153 pigment plays a minor role in ultrafast relaxation dynamics. Instead, the difference between APC and CPC arise primarily from pigment-protein interactions that modulate pigment geometry and vibronic structure. These results highlight the critical importance of local protein environments in controlling energy relaxation and coherence in photosynthetic light-harvesting proteins.