Quantum Effects and Light-harvesting: From the algal cell’s point of view

1. RC2 RC1 RC2 RC1 CP26 CP29 LHCII trimer LHCI LHCI FCPs RC2 RC1 Quantum Effects and Light-harvesting: From the algal cell’s point of view Beverley R. Green Botany Department, University of British Columbia, Vancouver, B.C., Canada

2. Light-harvesting from the algal cell’s point of view The BIG QUESTIONS: • How does it work? • How did it get that way? • Does it matter? Three views of the cryptophyte alga Guillardia theta Live cells (phase contrast microscopy) (Meriem Alami) SEM image (G. McFadden) Fluorescence confocal image showing “wrap-around” plastid (Yunkun Dang)

3. 1Chl* Light-harvesting antennas use only 3 types of chromophore! • Chlorophylls and bacteriochlorophylls • Absorb in blue and red/infrared regions. • Phycobilins - open-chain tetrapyrroles • Absorb 490-600 nm (green/yellow) • Carotenoids –absorb and protect • Harvest light but also convert energy from excited chlorophyll to heat - photoprotection heat (detected as fluorescence quenching (NPQ) Car The proteins make the difference--> huge variety of antennas B.R. Green (2001) “Was ‘molecular opportunism’ a factor in the evolution of different photosynthetic light-harvesting pigment systems?” PNAS 98: 2119-2121

4. Variety of Light-harvesting Antennas Plant s Cryptophyte algae Green sulfur bacteria Dinoflagellate algae Purple bacteria See “Light-harvesting Antennas in Photosynthesis” (2003) ed. B.R.Green, W.W. Parson.

5. Example #1: Purple bacterial LH2 antenna Ostroumov et al. Science (2013) 340, 52–56. Robert et al. (2003) Chap.4 in “Light-harvesting Antennas in Photosynthesis”, B.R.Green & W.W.Parson, Eds.

6. RC2 RC1 Example #2: Phycobilisome of cyanobacteria, red and glaucophyte algae An energy cascade to RC2 PEF575nm > PCF640nm > APCF660nm > LCMF682nm >Chl (RC2) reaction centre linker phycocyanin allophycocyanin phycoerythrin phycobilisome Chl PE PC APC Chll Absorbance Photosynthetic membrane 0 400 450 500 550 600 650 700 Wavelength (nm) Phycobilisome doesn’t bind carotenoids. But its efficiency is regulated by an orange carotenoid protein.

7. RC2 RC1 RC2 RC1 Lhcs RC2 RC1 Example #3: The LHC Superfamily: • Eukaryotic invention • No energy cascade • Pigments hung all over the place • Big protein family • Carotenoids -both for photoprotection and light-harvesting Plant LHCII Liu et al. (2004). Lhcs Lhcs “Brown” algae -Chl a/c Green algae and plants -Chl a/b Red algae-Chl a only

8. RC2 RC1  1 2  Cryptophytes-an amazing evolutionary story! Chloroplasts acquired by secondary endosymbiosis Chloroplast envelope (2 membranes) Nucleomorph N2 N2 N1 2nd Host (heterotroph) Cryptophyte Red alga Phycobilisome Photosynthetic membrane <-- NEW!! Gene loss Cryptophyte Red alga LHC RC2 RC1 Gene gain: new α subunit A brand new antenna from parts: β subunit from ancestral phycobilisome α subunit from…? Assembled tetramer relocated to thylakoid lumen NEW α’s

9. Anatomy of a cryptophyte phycobiliprotein α1 subunit (red) 9 kDa β subunit 19 kDa β subunit 19 kDa α2 subunit (blue) 8 kDa • Alpha subunits are the key: interactions between the α subunits and between α and β subunits hold the tetramer together. • They are the most variable in sequence. From Macpherson and Hiller (2003) in Light-harvesting Antennas in Photosynthesis (ed. B.R.Green, W.W. Parson). Based on x-ray structure of Wilk et al, PNAS 96: 8901-8906, 1999.

10. Cryptophytes also evolved a variety of new phycobilin pigments α subunit binds 1 phycobilin β subunit binds 3 phycobilins Result: great variety in wavelengths of light absorbed. PC 645 demonstrates electronic coherence PC645 from Chroomonas sp.

11. Light-harvesting from the algal cell’s point of view PC 645 from Chroomonas demonstrates electronic coherence So does PE545 from Rhodomonas Pair of DVB The two central bilins are in van der Waal’s contact and strongly coupled

12. Light-harvesting from the algal cell’s point of view X-ray Crystallography produced a surprise: Hemiselmis phycobiliproteins are different from all the others. (H. andersenii: PE555, H. virescens: PC612, H. pacifica: PC577) “Closed” Chroomonas-PC645 and all the others “Open” Hemiselmis-PC612, PE577, PE555 Hemiselmis-the two αβ units are rotated 73º with respect to each other, giving a hole down the middle (“open” configuration) and the central pair of pigments are no longer in contact. Harrop et al. (2014) Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins. PNAS Early Edition: www.pnas.org/cgi/doi/10.1073/pnas.1402538111

13. A single amino acid insertion (- charge) in the α subunit sequence is responsible for the open conformation! • Found only in Hemiselmis species ..(“open” form) • Causes a chain-reaction of rearrangements of amino acids….the two αβ units cannot fit closely together. • This mutation appears to have arisen within the Chroomonas clade and been amplified by gene duplication.

14. Strong excitonic coupling in closed form only Closed structures Open structures Harrop et al. (2014) Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins. PNAS Early Edition: www.pnas.org/cgi/doi/10.1073/pnas.1402538111.

15. 2D Electronic Spectroscopy shows electronic and vibrational coherences in closed form Closed Open Open Harrop et al. (2014) Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins. PNAS Early Edition: www.pnas.org/cgi/doi/10.1073/pnas.1402538111

16. John Archibald and Naoko Tanifugi, Dalhousie University, Halifax Marine Microbial Eukaryote Transcriptome Project Another twist to the story: “BIG DATA” changed the picture! • Hemiselmis species have genes for both “open” and “closed” α subunits! • Some of the “closed” form genes were as highly expressed as the “open” forms! • Cell used for mRNA isolation and sequencing were grown underHIGH LIGHT (100-150 mol m-2 sec-1) • Cells used for our targeted gene sequencing, crystallography and spectroscopy were grown under LOW LIGHT! (20-40 mol m-2 sec-1) This suggests that electronic coherence may play a part in ACCLIMATION to high light intensity by allowing synthesis of “closed” forms, ie. quantum switching. An elegant test: Will the "closed" form purified from an "open" species demonstrate electronic coherence? This test of the relationship between protein conformation and coherent behavior awaits funding.

17. The Cryptophyte Phycobiliprotein Consortium: Molecular biology and sequence analysis (Vancouver and Sydney, Australia) • Beverley Green • Chang Ying (Ivy) Teng • Roger Hiller Spectroscopy (Toronto) • Gregory Scholes • Tihana Mirkovic • Daniel Turner • Rayomond Dinshaw • Daniel Oblinsky Crystallography (Sydney, Australia) • Paul Curmi • Stephen Harrop • Krystyna Wilk • Roger Hiller Genomics/transcriptomics (Halifax) • John Archibald • Naoko Tanifugi • Dr. Kerstin Hoef-Emden (Köln) published a key paper relating cryptophyte evolution and phycobiliproteins (J. Phycology 44: 985-993, 2008), and provided cultures and much advice. We gratefully thank DARPA for funding research on cryptophyte photosynthesis! • Thanks also to the Australian Research Council, the Natural Sciences and Engineering Research Council of Canada, the Moore Foundation and DOE-JGI Eukaryotic Genomics.

18. Guillardia theta (cryptophyte) Thalassiosira pseudonana (diatom) Aureococcus Anophagefferens (brown tides) Fragilariopsis cylindrus (diatom) Phaeodactylum tricornutum (diatom) Emiliania Huxleyii (haptophyte) Biologists do it differently….. • High-throughput (aka brute force) methods, e.g. genome sequencing. • Mutant isolation—mutate first, ask questions later. • Relative (comparative) measures to understand an effect. Internal controls. • Bottom up (experiment to theory) as opposed to top down (theory to experiment)