Reconstitution of carotenoids in the light-harvesting 1 complex of Rhodospirillum rubrum for excited state dynamics studies

Abstract

During the primary events of photosynthesis, carotenoids function in important accessory light harvesting and photoprotective roles. In both oxygen evolving and anoxygenic phototrophs, these highly unsaturated polyenes are found in light-harvesting (LH) proteins where their energy transfer and excited state deactivation pathways have become active areas of investigation. Foremost among unresolved questions is the role played by the protein scaffolding in conferring functionally crucial conformational constraints that modulate these unique photophysical properties. Their LH activity is mediated by two singlet-excited states: S₂, accounting for their strong blue-green light absorption, and S₁, accessible only through decay of S₂. In LH complexes from anoxygenic phototrophic bacteria, a unique S₂ decay channel, designated as S^*, that promotes direct, ultrafast carotenoid triplet formation, has been discovered. In carotenoids of sufficiently high S₁ energy, S^* also functions in a new energy transfer pathway to bacteriochlorophyll α (BChl). In the Rhodospirillum rubrum LH1 complex, containing spirilloxanthin in a twisted configuration, the energy of the S₁ state is too low to transfer energy to BChl, and the triplet yield from the S^* state is unusually high (~30%). The triplet state is thought to be generated directly by fission of the S^* state into a pair of triplets localized on separate halves of the polyene chain, as governed by protein-induced polyene chain distortion. To test whether this protein binds other carotenoids in a twisted conformation and alters their excited state dynamics, the Rs. rubrum LH1 protein in a carotenoidless form was reconstituted with neurosporene, a carotenoid of shorter conjugation length. The reconstituted carotenoid was found to occupy most of the potential carotenoid binding sites and showed an absorption redshift characteristic of protein-bound polyenes. The strength of the carotenoid association was confirmed in a photoprotection assay (t_½ of BChl Q_y absorption band of neurosporene reconstituted LH1 = 203 min, vs. 238 min in wild-type, and 17.5 min in carotenoidless LH1 proteins). Preliminary ultrafast transient absorption measurements showed that the reconstituted complex transferred some energy from the S₁ state to BChl and also generated a neurosporene triplet state. Future studies will involve a more complete characterization of excited state dynamics, determination of the conformation assumed by the incorporated carotenoid, and reconstitution of carotenoids of other conjugation lengths.