A photosynthetic antenna complex foregoes unity carotenoid-to-bacteriochlorophyll energy transfer efficiency to ensure photoprotection Dariusz M. Niedzwiedzkia,b,1, David J. K. Swainsburyc, Daniel P. Canniffed, C. Neil Hunterc, and Andrew Hitchcockc,1 aCenter for Solar Energy and Energy Storage, Washington University in St. Louis, St. Louis, MO 63130; bDepartment of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130; cDepartment of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom; and dInstitute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom Edited by Krishna K. Niyogi, University of California, Berkeley, CA, and approved February 6, 2020 (received for review November 27, 2019) Carotenoids play a number of important roles in photosynthesis, pigment composition reduces the number of features that must primarily providing light-harvesting and photoprotective energy be interpreted in spectroscopic analyses. dissipation functions within pigment–protein complexes. The car- Numerous studies have shown that the Car-to-BChl a energy – = bon carbon double bond (C C) conjugation length of carotenoids transfer efficiency (ΦCar → BChl) varies in LH2s from different (N), generally between 9 and 15, determines the carotenoid-to- purple bacterial species and appears to correlate with the Car (bacterio)chlorophyll [(B)Chl] energy transfer efficiency. Here we pu- conjugation length. The yields for Cars such as neurosporene rified and spectroscopically characterized light-harvesting complex (N = 9) or spheroidene (N = 10) are 85 to 95% (11, 19, 23–25). 2 (LH2) from Rhodobacter sphaeroides containing the N = 7ca- The yields decrease to 55 to 65% for Cars where N = 11 (e.g., rotenoid zeta (ζ)-carotene, not previously incorporated within rhodopin, rhodopin glucoside, or lycopene) (11, 24–26) and re- a natural antenna complex. Transient absorption and time-resolved duce further to ∼30% when N = 13 (spirilloxanthin) (14). As fluorescence show that, relative to the lifetime of the S1 state of longer C=C conjugation length (larger N) lowers the energies of ζ-carotene in solvent, the lifetime decreases ∼250-fold when both the S and S excited states (2, 6), the broadly accepted ζ 2 1 -carotene is incorporated within LH2, due to transfer of excitation explanation for the variation in Φ → in LH2 is linked with a Car BChl energy to the B800 and B850 BChls . These measurements show the inherent excited-state properties of the particular Car. Typ- ∼ that energy transfer proceeds with an efficiency of 100%, primar- ically, both S and S states are involved in energy transfer to → → 2 1 ilyviatheS1 Qx route because the S1 S0 fluorescence emission of BChls, and if the S state falls below the Q bands of the B800 ζ 1 y -carotene overlaps almost perfectly with the Qx absorption band of and B850 spectral forms of BChl a, reducing spectral overlap of the BChls. However, transient absorption measurements performed on the hypothetical S1 emission and BChl a absorption, then the microsecond timescales reveal that, unlike the native N ≥ 9 carotenoids ΦCar → BChl declines sharply. At first glance, some Cars do not normally utilized in light-harvesting complexes, ζ-carotene does not N = a appear to fit into this correlation, for example, okenone with quench excited triplet states of BChl , likely due to elevation of the 13 + C=O, where nominally there are 13 conjugated carbon–carbon ζ-carotene triplet energy state above that of BChl a. These findings provide insights into the coevolution of photosynthetic pigments and pigment–protein complexes. We propose that the N ≥ 9 caroten- Significance oids found in light-harvesting antenna complexes represent a vital compromise that retains an acceptable level of energy trans- Photosynthesis uses carotenoids as light-harvesting pigments fer from carotenoids to (B)Chls while allowing acquisition of a and for photoprotective energy dissipation. The carbon–carbon new, essential function, namely, photoprotective quenching of double bond conjugation length of carotenoids (N) affects the harmful (B)Chl triplets. carotenoid-to-(bacterio)chlorophyll energy transfer efficiency, but the photoprotective capability was considered to be in- photosynthesis | carotenoids | light-harvesting | photoprotection | ultra- dependent of N. Using light-harvesting complex 2 from the fast spectroscopy model photosynthetic bacterium Rhodobacter sphaeroides con- taining ζ-carotene (N = 7) or neurosporene (N = 9), we dem- onstrate that decreasing the conjugation length increases the arotenoids (Cars) are natural pigments found in plants, al- carotenoid-to-bacteriochlorophyll energy transfer efficiency, in gae, and photosynthetic (cyano)bacteria where they function C the case of ζ-carotene to ∼100%. However, unity quantum ef- in light-harvesting, stabilization of pigment–protein complex ficiency comes at the cost of photoprotection, suggesting that structure, and photoprotection (1, 2). In antenna complexes and naturally evolved photosynthesis tolerates some energetic loss photosystems, Cars act as accessory light-harvesting pigments to allow essential energy dissipation, explaining why longer- and transfer excitation energy to (bacterio)chlorophylls [(B) conjugation length carotenoids are utilized in native pigment– Chls]. The absorption bands of Cars typically span the 400 to protein complexes. 550 nm spectral range and are associated with the S0 → S2 – electronic transition of the linear carbon carbon double bond Author contributions: D.M.N. and A.H. designed research; D.M.N., D.J.K.S., D.P.C., (C=C) conjugation (N), a characteristic structural motif (3, 4). and A.H. performed research; D.M.N., D.J.K.S., D.P.C., C.N.H., and A.H. analyzed data; Cars also serve important photoprotective functions in light- and D.M.N., D.J.K.S., D.P.C., C.N.H., and A.H. wrote the paper. harvesting complexes, typically quenching harmful excited trip- The authors declare no competing interest. let states of (B)Chls to inhibit formation of reactive oxygen This article is a PNAS Direct Submission. species such as singlet oxygen (5). The photophysical properties Published under the PNAS license. of Cars and their roles in numerous light-harvesting antennas 1To whom correspondence may be addressed. Email: [email protected] or have been investigated (6). The LH2 complex from purple [email protected]. photosynthetic bacteria has been a model antenna for such This article contains supporting information online at https://www.pnas.org/lookup/suppl/ studies (7–16) as crystal structures are available (17, 18), the Car doi:10.1073/pnas.1920923117/-/DCSupplemental. content can be manipulated (14, 19–22), and its relatively simple First published March 5, 2020. 6502–6508 | PNAS | March 24, 2020 | vol. 117 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1920923117 Downloaded by guest on October 1, 2021 bonds and a terminal C=O group that could extend the conjugation length. Okenone bound within the LH2 complex from the bacterium Merichromatium purpuratum (formerly Chromatium purpuratum) shows ΦCar → BChl = ∼86% (27), substantially above the expected value of ∼30% observed for other N = 13 Cars. However, previous investigations have demonstrated that when conjugation is extended into the cyclic ends of Cars, the double bonds in the rings act as only ∼0.3 of a double bond in the linear backbone, and in case of Cars with aryl rings such as okenone the contribution may be even smaller (28, 29). Indeed, it has been shown that the energies of the excited states of okenone are only marginally smaller than their counterparts for the open chain Car spheroidenone (N = 10 + C=O) (30), which when bound to LH2 shows a ΦCar → BChl of 92 to 94% (11, 20). Unlike ΦCar → BChl efficiency, the ability to provide photo- protective quenching of BChl a excited states (triplets) is more or less the same for all of the above mentioned Cars incorporated into LH2 (7, 31, 32). It could therefore be perceived that the Fig. 1. Spectroscopic comparison of the ζ-carotene–containing LH2 antenna ability to photoprotect (B)Chls in photosynthetic antennas is a complex from R. sphaeroides and all-trans-ζ-carotene in solvent at room universal characteristic of all Cars; however, this is not the case. temperature. The chemical structure of all-trans-ζ-carotene is shown above Energetic requirements between (B)Chl and Car triplet states the panels. The C=C bonds highlighted in red indicate the conjugation length (N = 7) of the Car. (A) Comparison between absorptance (black) and must also be considered, and, like the S2 and S1 states, the triplet ζ states of Cars have well-defined energies that also depend on N, fluorescence excitation (red) spectra shows that, in LH2, -carotene transfers excitation energy to BChl a essentially without any energetic loss. (B) All- although this is impossible to confirm directly due to undetect- trans-ζ-carotene dissolved in solvent shows pronounced fluorescence emis- able phosphorescence. Interestingly, photosynthetic organisms sion from the S1 state and a much weaker emission band from the S2 state. have a strong preference for Cars with N ≥ 9 for incorporation into light-harvesting proteins (1), suggesting that these Cars have triplet state energies in a sweet spot corresponding to the triplet of ζ-carotene) (10); therefore, it is possible that the majority of state energies of specific (B)Chls to ensure the most efficient the ET occurs from the S1 excited state. It is broadly accepted sensitization [(B)Chl triplet quenching] (33). In support of this that LH2 complexes bind Cars in all-trans geometry (17), and for BIOCHEMISTRY idea, experiments that tested photoprotective abilities of open comparative studies with isolated Car in solvent, we purified the chain and cyclic Cars of different N lengths in Car–Chl a model all-trans geometric isomer of ζ-carotene (the purification mixtures demonstrated that photoprotective capability appears procedure is described in SI Appendix,Fig.S2). To examine N ≤ – to fall sharply for Cars with 8 (34 36).