Structural and spectroscopic basis of excitation energy transfer in microbial rhodopsins binding xanthophylls

Carotenoids serve as accessory light-harvesting pigments in microbial rhodopsins, but the mechanisms enabling efficient energy transfer in systems lacking canonical 4-keto groups remain poorly understood. Here, we combine long-timescale molecular dynamics, polarizable quantum mechanics/molecular mechanics (QM/MM) calculations, and excitonic modeling to elucidate the structural and electronic factors that govern carotenoid-to-retinal excitation energy transfer (EET) in the proton-pumping rhodopsin Kin4B8. Focusing on the xanthophylls, zeaxanthin and lutein, we show they support ultrafast (<100 fs) and high-efficiency (≈70%) EET, enabled not by specific functional groups but by precise protein-ligand geometry. The carotenoid's β-ring is anchored <i>via</i> a dynamic hydrogen-bonding network with Ser208 and Tyr209 within a conserved protein cavity, a configuration that optimally positions the retinal and carotenoid chromophores for strong excitonic coupling. Simulated absorption and circular dichroism (CD) spectra accurately reproduce observed spectral features, including the characteristic biphasic CD band shapes, notably the blue-shifted CD minimum compared to the absorption peak in the retinal region. A Förster-type kinetic model, built from QM/MM-derived parameters, recovers experimental transfer times and efficiencies. Our findings provide a mechanistic rationale for recent mutagenesis and carotenoid screening experiments, establishing that rhodopsin-based light harvesting is driven by protein-guided chromophore alignment rather than fixed carotenoid chemistry. This work establishes design principles for engineering photoactive proteins and offers a transferable framework for analyzing energy transfer across natural and synthetic light-harvesting systems.