Structural inhomogeneity and inter-chromophore electronic/protein-mediated coupling in the Gloeobacter...

Microbial rhodopsins are photoreceptive membrane proteins that utilize retinal as a chromophore to absorb light and drive diverse photochemical functions. Recent studies have revealed that some microbial rhodopsins bind carotenoids in addition to retinal. The bound carotenoids may function as light-harvesting antennas in vivo by absorbing light in spectral regions where retinal absorbs less efficiently and transferring the excitation energy to the retinal chromophore. Here, we employed resonance Raman spectroscopy with excitation wavelengths from 440 to 600 nm to investigate the retinal and carotenoid chromophores in the complex of Gloeobacter rhodopsin (GR) and canthaxanthin (CAN). We found that the binding of one chromophore to the protein affects the π-conjugated system of the other chromophore, indicating mutual perturbation between retinal and CAN. We also observed excitation-wavelength-dependent shifts in the ν(C=C) vibrational bands of both retinal and CAN, providing evidence for structural inhomogeneity of the chromophores. Model calculations supported the presence of multiple substates within the ensemble. These findings provide new spectroscopic insights into interchromophore interactions and structural heterogeneity in the GR-CAN complex. Moreover, this study demonstrates that variation of the resonance Raman excitation wavelength provides a structurally sensitive means of probing intrinsic inhomogeneity within ensembles of protein-bound chromophores.