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

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

A quenching mechanism mediated by the orange carotenoid protein (OCP) is one of the ways cyanobacteria protect themselves against photooxidative stress. Here, we present a femtosecond spectroscopic study comparing OCP and RCP (red carotenoid protein) samples binding different carotenoids. We confirmed significant changes in carotenoid configuration upon OCP activation reported by Leverenz et al. (Science 348:1463-1466. doi: 10.1126/science.aaa7234 , 2015) by comparing the transient spectra of OCP and RCP. The most important marker of these changes was the magnitude of the transient signal associated with the carotenoid intramolecular charge-transfer (ICT) state. While OCP with canthaxanthin exhibited a weak ICT signal, it increased significantly for canthaxanthin bound to RCP. On the contrary, a strong ICT signal was recorded in OCP binding echinenone excited at the red edge of the absorption spectrum. Because the carbonyl oxygen responsible for the appearance of the ICT signal is located at the end rings of both carotenoids, the magnitude of the ICT signal can be used to estimate the torsion angles of the end rings. Application of two different excitation wavelengths to study OCP demonstrated that the OCP sample contains two spectroscopically distinct populations, none of which is corresponding to the photoactivated product of OCP.

• Klíčová slova
• Intramolecular charge-transfer state, Non-photochemical quenching, Orange carotenoid protein, Red carotenoid protein, Ultrafast spectroscopy,
• MeSH
• karotenoidy analýza   MeSH
• sinice chemie   MeSH
• spektrální analýza metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• karotenoidy MeSH