Structural and Spectroscopic Basis of Excitation Energy Transfer in Microbial Rhodopsins Binding Hydroxylated Carotenoids

Rhodopsin–carotenoid complexes are emerging as versatile light-harvesting systems that expand microbial phototrophy beyond the intrinsic absorption range of retinal. While 4-ketocarotenoids have traditionally been considered essential for excitation energy transfer (EET) in these systems, recent experimental discoveries have demonstrated that hydroxylated carotenoids, such as zeaxanthin and lutein, can also mediate efficient EET, despite lacking keto groups. Here, we elucidate the molecular basis for this phenomenon in the Kin4B8-xanthorhodopsin using a multiscale computational approach combining molecular dynamics, polarizable quantum mechanics/molecular mechanics (QM/MM) calculations, and excitonic modeling. We identify four key features underlying efficient energy transfer: (i) both zeaxanthin and lutein bind preferentially via their β-rings in a conserved protein fenestration, stabilized by hydrogen bonds with Ser208 and Tyr209; (ii) enhanced sampling simulations confirm that the fenestration defined by Gly153 is essential for carotenoid binding and energy transfer, as shown by functional loss in G153F mutant; (iii) strong excitonic coupling (250–300 cm−1) between the carotenoid S2 and retinal S1 states drives ultrafast EET with ∼70% efficiency and sub-100 fs transfer times, matching experimental data; (iv) the biphasic circular dichroism spectrum originates primarily from excitonic coupling rather than induced chirality. Our findings explain how structural complementarity, rather than specific carotenoid functionality, governs light-harvesting capability in rhodopsin–carotenoid complexes, providing mechanistic insight and design principles for engineering biomimetic phototrophic systems.