Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• fotochemie * MeSH
• karotenoidy metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie MeSH
• molekulární modely MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• karotenoidy MeSH
• orange carotenoid protein, Synechocystis MeSH Prohlížeč

The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.

• Klíčová slova
• OCP1, OCP2, Photoactivation, Ultrafast spectroscopy, X-ray footprinting,
• MeSH
• bakteriální proteiny chemie   MeSH
• fluorescenční spektrometrie MeSH
• fykobilizomy chemie   MeSH
• sinice chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fykobilizomy MeSH

RC-LH1-PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC-LH1-PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S2-Qx pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC-LH1-PufX we observe an additional, minor energy transfer pathway associated with the S1 state of diketospirilloxanthin. By comparing the spectral properties of the S1 state of diketospirilloxanthin in solution, in LH2, and in RC-LH1-PufX, we propose that the carotenoid-binding site in RC-LH1-PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S1/ICT-mediated energy transfer channel.

• Klíčová slova
• Carotenoids, Energy transfer, Intramolecular charge transfer state, Light-harvesting, Purple bacteria, Ultrafast spectroscopy,
• MeSH
• bakteriochlorofyly metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• karotenoidy metabolismus   MeSH
• kinetika MeSH
• počítačové zpracování signálu MeSH
• přenos energie * MeSH
• Rhodobacter sphaeroides metabolismus   MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• xanthofyly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriochlorofyly MeSH
• karotenoidy MeSH
• spirilloxanthin MeSH Prohlížeč
• světlosběrné proteinové komplexy MeSH
• xanthofyly MeSH