A design principle for tuning far-red absorption of chlorophyll a in light-harvesting complexes

Far-red absorption in eukaryotic light-harvesting complexes (LHCs) has been associated with strongly excitonically coupled chlorophyll a clusters exhibiting mixing with charge-transfer states, yet the structural rules enabling this spectral tuning remain unclear. Previous studies have highlighted the importance of the amino acid ligating Chl a603 in providing the pigment orientation required for the formation of a red-shifted Chl a603-a609 cluster. More recently, it has been suggested that the steric properties of the residue at the i-4 position from the ligand may also play a crucial role.Here, we test this hypothesis through targeted mutagenesis of two light-harvesting complexes, Lhca4 and CP29, which host the Chl a603-a609 pair, but differ in their protein environment and spectral properties. In Lhca4, introduction of steric constraints at the i-4 position relative to the Chl a603 ligand (A43L) abolishes far-red absorption, indicating that steric crowding at this position destabilizes the strongly coupled pigment configuration. In CP29, substitution of the Chl a603 ligand (H111N) is required for far-red absorption, while the additional mutation at i-4 position (H111N/C107A) modulates the magnitude of the red-shift. Together these results highlight the importance of both axial ligand identity and local protein environment in controlling far-red absorption in Chl a clusters.