Cryospectroscopy Studies of Intact Light-Harvesting Antennas Reveal

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Cryo-Spectroscopy Studies of Intact Light-Harvesting Antennas Reveal Empirical Electronic Energy Transitions in Two Cyanobacteria Species Albert Collins Nganou Assonkeng, Noam Adir, and Martin Mkandawire J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00714 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

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Cryo-spectroscopy studies of intact light-harvesting

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antennas reveal empirical electronic energy

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transitions in two cyanobacteria species

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Collins Nganou,† Noam Adir § and Martin Mkandawire†,*

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†

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P.O. Box 5300, 1250 Grand Lake Road, Sydney, Nova Scotia, Canada B1P 6L2

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§

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* Corresponding author: Tel. +1-902-563-1430; Fax. +1-902-563-1360; Email:

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[email protected].

Verschuren Centre for Sustainability in Energy and the Environment, Cape Breton University,

Schulich Faculty of Chemistry, Technion, Israel Institute of Technology, Haifa, 32000 Israel

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1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT.

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Understanding of electronic energy transition (EET) mechanisms from the light-harvesting unit

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to the reaction centre in a natural system has been limited by; (a) the use of conventional

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transient time-resolved spectroscopy at room temperature, which result in high signal-to-noise

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ratio; and (b) examining extracts instead of intact light-harvesting units. Here, we report

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previously unknown differences and new insight in EET of two cyanobacteria species,

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Acaryochloris marina and Thermosynechoccocus vulvanus, which have been found only after

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using Uv-vis, hole-burning and fluorescence spectroscopy at ultra-low temperature and

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examining their intact light-harvesting unit, phycobilisomes (PBS). Although the exciton

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formation is similarly induced by photoexcitation of chromophore assemblies in phycocyanin

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(PC) and allophycocyanin (APC) in PBS’s of both species, the EET mechanisms are totally

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different, being adiabatic in A. marina and non-adiabatic in T. vulvanus. The PBS of A. marina

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has only one APC trimer and energy transfer is through coupling of α84 in APC with β84 in

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adjacent PC. In T. vulvanus, the PBS has three components: coupling between APC core and the

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entire PC rod; couplings of β-β18 and of LCM to β in the adjacent APC-like trimer. 80% of the

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excitation energy is trapped in the coupling β-β18 and regulates the flow of energy from the high

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to low-level terminal electronic transition emitter β-LCM. All these details cannot be observed at

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room temperature and in extracted units.

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INTRODUCTION

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Development of technologies based on mimicking of biological systems is gaining prominence

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due to expectation that such technologies would be as efficient and clean as nature is.

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Consequently, there has been surge in studies of natural systems functioning in the last two

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decades, which have resulted into emerging of bio-inspired designs of devices on the market.

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Bio-inspired designing of devices requires thorough understand of processes in a system being

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mimicked. For instance, improved harvesting and conversion efficiency of solar radiation to

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other forms of energy through bio-mimicry would requires a good understanding of biophysical

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and photochemical processes involved in photosynthesis, especially of efficient photosynthetic

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organism like cyanobacteria. Cyanobacteria have one of most advance photosynthesis process,

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which enabled them to adapt to low light intensity environments. They use phycobilisomes

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(PBS) as primary light-harvesting antenna, which are capable to harvest sunlight in the spectrum

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gap that is normally not absorbed by chlorophyll (Chl) in higher photosynthetic organism. 1-6

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The PBS is composed of protein, which binds pigments such as bilins (called cofactor) in a

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specific position. Upon absorption of light, the pigments in PBS are excited and deliver the

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excitation energy to the photosynthesis reaction centre (RC). It is not yet unanimously agreed on

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how the PBS is attached to RC and how the attachment is structurally organized.1, 3, 7-9 Generally,

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the PBS is consist of protruded array of rods around the allophycocyanin (APC) core, which may

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attach to the RC of photosystem II (PS II) via a pigmented linker core membrane (LCM). Recent

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studies reported some cyanobacteria species, for instance Anabaena sp. PCC 7120, with the PBS

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attaching to the RC of photosystem I (PS I).7 Unlike in thermophilic cyanobacterium

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Thermosynechoccocus vulcanus, the PBS in Anabaena sp. PCC 7120 is linked to the PS I

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without APC core.1, 7 This is evidenced by fluorescence at 665 nm, attributed to phycocyanin 3 ACS Paragon Plus Environment


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(PC) hexamer binding to PS I via CpcG3 (named CpcL), a pigmented linker protein.10 Therefore,

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the inhomogeneous broader absorption and emission of the PC event at 77K does not give a clear

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picture of fluorescence and absorption transition, thereby requiring further investigations on the

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electronic transition of PC in intact PBS at the 665 nm.

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The PBS are located on the stromal surface of the thylakoid, and their building blocks are α

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and β polypeptide (open tetrapyrrole cofactors) subunits. The subunits combine to form a

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monomer, which each covalently bind at least two bilin cofactors. The assembly of open

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tetrapyrrole cofactors and linker proteins in the PBS is responsible for the sub-complex

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functional absorption between 500 and 680 nm. In general, the PC monomers in PBS contain

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three phycocyanobilins (PCBs); two bound to the conserved cysteines at position 84 on each

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subunit and one to a cysteine at position 155 of the β subunit. Monomers of APC, the phycobilin

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protein (PBP) found in the core of PBS, contain only two cofactors at the position 84.8 The PC

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trimer has its absorption maximal at 620 nm at RT,10 while in the isolated PC rod of the T.

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vulcanus has the maximum absorption at 635 nm at RT.10 In T. vulcanus, the APC core and

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trimer have maximum absorption around 652 nm.

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In cyanobacterium Acaryochloris marina, the PBS antenna complex has a molecular weight of

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about 1.2 MDa and its APC trimer has maximum absorption around 640 nm at RT. The resonant

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absorption maxima of the individual bilin cofactors β155, α84, β84 in the PC monomer assembly at

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77K are around 600–602, 624 and 628–630 nm, respectively.11 Isolated A. marina PBS and even

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its isolated trimers contain mixtures of two isoforms of the α subunit and two of the β subunit. In

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purified PC solution, there are about three times as much of β1 as β2 units, while the ratio of α1

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to α2 is about 3:2. This arrangement must have some effect on the EET between rods, and

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perhaps to the RC as well. APC is hardly seen in these isolated PBS, which suggest that there is


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either only a single APC monomer in the terminal trimer (i.e. two PC monomers and one APC

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monomer), or only a relatively few whole PBS (tetra-hexamers) have the terminal PC/APC

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heterohexamer. At both low temperature, highly purified PC emits at 656-659 nm, red-shifted

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compared to T. vulcanus. Since it was suggested that the A. marina APC is blue shifted

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compared to other APCs, this brings them into the same ballpark. What this may mean is that

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some “APC” might be red-shifted PC.

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In a recent study, Tiwari and co-workers have suggested that anticorrelated component of

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pigment vibrations can drive non-adiabatic electronic energy transfer.2 They further suggest that

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the route of efficient energy is resonantly enhanced through Franck Condon (FC) vibrational

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mode of the monomeric pigment.2 However, the signal of the exciton mode activity was

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significantly higher at 19 K12 than 80 K2, suggesting existence of reasonable trapping of the

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thermal fluctuation. This trapping of thermal fluctuation may enhance the active signal of the

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electronic coupling. In this framework, the average energy gap between excitons was expected to

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be within the pigment vibrational frequency in the fluorescence spectrum because of their

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resonance mechanism activity.2 Thus, reducing the thermal fluctuation would help to minimise

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all the interference and lead to a clear signal that would enhance the understanding of electronic

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transition in intact PBS.

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Consequently, we investigated and compared two antenna systems – the PBS of T. vulcanus

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and A. marina – at 4 K. At this low temperature, the thermal fluctuation is expected to be

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extremely reduced and all electronic transition to be in resonant mode activity after excitation.

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The interest to compare the systems was based on the difference in the PBS architectural

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organization of the species (Scheme 1): where it is organized as a single rod-shaped assembly of

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PBPs with the APC trimer in A. marina;13,14 and arranged as a pentacyclic core in which an array



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of protrusions of at least six PC rods in T. vulcanus. Therefore, it was hypothesised that using

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cryo-spectroscopic investigations of intact PBS set-ups would reveal the real-time electronic

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transition and mechanism, enabling possible to monitor the functional electronic sites of the PBS

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sub-compounds, because there is low thermal fluctuation at 4K. Further, the electronic coupling

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transition of the inter sub-complex between an intact PBS’s of A. marina and T. vulcanus would

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be different because of their differences in the PBS structural organizatioin. (A)

(B) PC Trimer Linker protein PC-PC

Linker protein PC-APC Linker protein PC-PC

PC Trimer PC620 PC612 Linker protein PC-PC PC Hexamer

PC Trimer Linker protein PC-APC

APC

APC Trimer Linker core membranes (LCM)

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Scheme 1. Arrangement of the PBS in (a) A. marina based on Chen et al. 14 and (b) T. vulcanus

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based on David et al.1

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EXPERIMENTAL AND THEORETICAL METHODS

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Acaryochloris marina (strain MBIC 11017; Technische Universität Berlin, Germany) were

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cultured at 26°C in seawater-based K medium,15 bubbled with filtered air under continuous

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shaking and white fluorescent lamp illumination14 (20 μmol photons m−2 s−1). Isolation of A.

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marina PBP antenna complexes were done as described by Marquardt et al.13 and Hu et al.16 In

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brief, A. marina cells were harvested from the culture by centrifugation; washed in potassium

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phosphate buffer (0.75M, pH 7.0) containing sucrose (10%), sodium ethylendiamine tetraacetic

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acid (2 mM) and phenylmethylsulfonyl fluoride (0.1 mM), shaken vigorously with glass beads (1 6 ACS Paragon Plus Environment

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mm diameter) in a cooled cell homogenizer for 15 min to break the cells. The glass beads were

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removed by filtration, and the non-soluble cell fractions centrifuged (47,000 g for 1 h). The pellet

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was suspended in potassium phosphate buffer containing N,N-dimethyldodecylamine-yV-oxide

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(1.5% final concentration) and stirred (40 min at 17°C) in the dark to solubilize the thylakoid

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membranes. Residual material was removed by centrifugation and the lysate loaded on sucrose

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gradients (15-35% w/v) in potassium phosphate buffer, centrifuged (100,000 g for 20 h) and the

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separated coloured bands harvested and subjected to further investigation.

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Common Thermosynechococcus vulcanus (strain RKN, Technion Haifa, Israel) were cultured

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at 45 °C in BG11 medium under continuous white fluorescent lamp illumination (30 μmol

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photons m−2 s−1) and bubbled with air containing 1.0% (v/v) CO2. To isolate T. vulcanus PBS, 12

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litres of harvested cells were re-suspended in phosphate buffer (0.9 M pH 7.0), disrupted with

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one French pressure cell treatment (20,000 psi), centrifuged (27000 g) and the pellet re-

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suspended in phosphate buffer, incubated (1 h with 2% Triton X -100 (w/v, Sigma)), and

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clarified with centrifugation. The supernatant was transferred to 10 ml tubes, ultracentrifuged (2

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h at 150,000 g Beckman Coulter, optima L-90 K ultracentrifuge, Beckman Type T70.1 rotor) and

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the blue pellet re-suspended in phosphate buffer, placed on a sucrose cushion (0.8 M),

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centrifuged (2 h at 190,000 g) and the pellet re-suspended in phosphate buffer, placed on a two-

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step sucrose gradient (1 M and 1.2 M in 0.9 M phosphate buffer), ultracentrifuged (overnight at

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150,000 rpm) and the three different bands isolated were characterized by absorption (Varian

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spectrophotometer—Cary Bio 50) and fluorescence spectroscopy (Flourolog, excitation at 580

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nm, with slit width of 5 nm for excitation and 1 nm for emission).

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The isolated intact T. vulcanus PBS were examined at low temperature (4 K). Glycerol (70%)

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was added to obtain good optical density before measuring absorption and fluorescence (Spectra



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Physics model 375 dye laser, line width of < 0.5 cm-1, pumped by an Ar-ion laser model 171,

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Spectra Physics, USA). The samples were transferred to plastic cuvettes placed in He-bath

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crysostat (Utreks, Ukraine) and the optical path (8 mm) was above the level of liquid He (4.2 ±

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0.2 K). The spectra were recorded by spectrograph (0.3 m spectrograph Shamrock SR-303i,

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Andor Technology, UK), equipped with an electrically cooled CCD camera (charged coupled

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device camera DV420A-OE, Andor Technology, UK).

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RESULTS AND DISCUSSION

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Photoreactions PBS pigment complexes in real-time. The exciton energy transition in intact

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light harvesting antenna, PBS, of the cyanobacteria, was probed under cryogenic temperature to

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reduce noise. Figure 1 presents electronic transition of A. marina and T. vulcanus measured at

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cryogenic temperature (i.e., 4 K). In an earlier publication, we presented electronic transition of

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A. marina and T. vulcanus measure at room temperature (i.e., 273 K).15 Generally, transient

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spectroscopy is a powerful tool of probing of photophysical and photochemical reactions in real

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time after light absorption by photosynthetic pigment–protein complex. However, probing intact

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light harvesting antenna, PBS, of cyanobacteria has been a challenge because the transient

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spectroscopy involves promoting a fraction of the molecules to an electronically excited state

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with an excitation (or pump) pulse at room temperature; and, the PBS has other molecules,

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which get excited together obscuring the electron transition signal. The excitation is broad in

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time and electronic states are excites.

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Pronounced resonant absorption peak on the two cyanobacteria PBS is observed (Figure 1a).

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The resonant absorption band observed around 600 nm was earlier shown by Debreczeny and co-

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workers to be induced by a β155 cofactor in monomeric PC.11 This is in presence of the hexameric


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assembly in the two-antenna system. The absorption spectrum of the β155 in monomeric PC11 and

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the resonant peak around 600 nm in the hexameric assembly of the PBS of A. marina and T.

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vulcanus indicate conservation of the β155 absorption. Further similarity between monomeric and

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hexameric PC assembly is fairly observed at 630 nm in the PBS of A. marina. This band has also

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been shown to be induced by the β84 in monomeric PC.11 In the PC absorption spectrum of the

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PBS of T. vulcanus, a shoulder appears at 630 nm and a further sharp absorption electronic peak

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forms at 635 nm. The shoulder at 630 nm and the 635 nm high absorption peak in PC of T.

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vulcanus are two distinct states. The 630 nm is an already known transition of the β84 cofactor.

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The 635 nm might be the low energy of the localized electronic transition of an exciton state

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with APC trimer, which was earlier described by McCall.16

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Figure 1. A steady state absorption comparison between PBS of A. marina in black and T.

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vulcanus in red at 4 K: (a) Absorption spectrum but with second derivative for PBS absorption of

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A. marina; and (b) comparison second derivative for PBS absorption of A. marina and T.

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vulcanus. A 10  1 nm bleu-shift of the PBS of A. marina is observed.

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Additionally to the absorption of β cofactor in PC of PBS of both A. marina and T. vulcanus,

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two distinct shoulders are observed around 550 and 575 nm. So far, there are neither biochemical 9 ACS Paragon Plus Environment


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nor fluorescence evidence of phycoerthrin (PE), which can be attributes to the shoulders, in the

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two antenna systems. Thus, the origin of the shoulders at 550 and 575 nm needs further

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investigation. The APC in PBS of T. vulcanus has absorption resonant peak at 653 nm, while the

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absorption of APC in the PBS of A. marina appears with a shoulder at 643 nm; vis-à-vis

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revealing a 101 nm red-shifted in PBS of T. vulcanus. This weak resonance absorption

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observed in the PBS of A. marina may be due to the unique set-up in which there is only one

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APC trimer in the last hetero-hexamer in its PBS, while there is a pentacyclic APC core in the

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PBS of T. vulcanus (see also Scheme 1). Earlier, Hu and co-workers showed that isolated APC

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from the PBS of A. marina has a maximum absorption at 640 nm at room temperature (RT).17

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Therefore, the 3 nm shift in between the result of Hu and co-workers and ours is due to the low

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temperature used in our study that reduced the thermal fluctuation, responsible for an

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inhomogeneous broader absorption of the cofactor in the protein matrix at RT.

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Furthermore, the PBS of T. vulcanus contains a PC612 near the APC core. To gain insight into

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the electronic absorption, the second derivative absorbance of these PBS’s were computed

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(Figure 1b). A spectrum with a plateau appears between 640 and 645 nm in the PBS of T.

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vulcanus. Knowing that the PC612 is the nearby sub-compound18 and that the linker PC rod-

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APC core does not have a bilin cofactor, this change was assigned to the electronic transition of

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PC612. The range of the plateau for the PBS of T. vulcanus spectrum appears at the same

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position where APC trimer in PBS of A. marina absorbs. There are two genes that encodes the

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PC in A. marina.1, 14, 19-21 Later, Adir found that PBS of T. vulcanus has one PC encoding gene.18

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Further, Adir showed that T. vulcanus has two different methylations of the Beta-N72 residue in

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PC620 and PC612.18 Despite having two genes encoding PC, the PBS of A. marina does not

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display two distinct absorption peaks, while the PBS of T. vulcanus with one encoding PC gene 10 ACS Paragon Plus Environment

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and the two different methylation of the Beta-N72 has a clear distinct signal on the second

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derivative in Figure 1(b). Therefore, the methylation is likely to be responsible for the different

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electron transition between PC620 and PC612, and not their genetics. When considering energy

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levels within the PC in PBS of T. vulcanus, there exist high and low energy levels induced by

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PC620 and PC612, respectively.

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Further, comparison between A. marina and PBS of T. vulcanus terminal pigment electronic

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absorption around 670‒680 nm displays an additional redshift of electronic absorption peak of

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the PBS of T. vulcanus. This complete bathochromic shift exhibited by the PBS of T. vulcanus

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may be facilitated by the structural assembly, where there is difference energy distribution due to

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having a PC612 between 640‒645 nm in the PC rod, and APC E1 LCM. However, the

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computation of the second derivative spectrum shows a close overlap spectrum of APC E

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electronic site. Therefore, a plausible explanation of bathochromic shift can perhaps be the losses

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of a fraction of the terminal emitter pigment in PBS of A. marina.

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Role of co-factor couplings in electronic transition. As observed in Figure 1a, the PBS of T.

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vulcanus exhibits a maximum peak at 635 nm. However, the PBS of A. marina is also an

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assembly of homogeneous PC hexamer except for the last hetero-hexamer containing PC trimer

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and APC trimer. We found in previous work that the PBS of A. marina has an intermediate

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transition state like an exciton at 635 nm.22 Armed with this information, we scanned the zero

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phonon line excitation at very low and constant fluence excitation. With this approach, all the

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transitions in resonance increase in amplitude change of the absorption (see Figure 2).



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Figure 2. Low fluence hole burning action spectra of the PBS of A. marina. The points represent

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the experiment data. The fit curve was obtained with sum of 4 Gaussian functions, with the best

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fit with 4 centres. Laser power density (fluence of 2 mJ/cm2). APC F is the electronic transition

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involved couplings of β-β18; and the LCM involved β-LM.

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A transition at 635 nm appears in both PBS of A. marina and T. vulcanus. However, this

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electronic transition state is a low absorption electronic state of the PC in sub-compound in the

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PBS of A. marina, while the low electronic absorption of the PC rod in PBS of T. vulcanus

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appears at 643 nm (also see second derivative, Figure 1(b)). These findings support a hypothesis

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presented earlier in a report by Nganou.22 Further, both A. marina and T. vulcanus have

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absorption peak at 660 nm, and a broad electronic transition spectrum from 655 to 665 nm.

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However, T. vulcanus has redshift transition between 660 – 665 nm in relation to A. marina,

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which has been previously reported to be induced by potential coupling involving β18 (encoded in

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APC F gene) in LCM with cofactors β84 in neighbouring timers (i.e., β18-β84) in T. vulcanus.23-25

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The influence of β18 on the fluorescence emission at 665 nm was elaborated by Kuzminov and

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co-workers, who investigated different mutants of Synechocystis sp. PCC 6803 engineered to be

240

deficient of PSI/PSII, PSI/PSII/APCD and PSI/PSII/APCF.23 They found that the transition at

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665 nm always involved β18. Further they found that the transition 680 nm involved the coupling

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of LCM (encoding APC E and the once encoding APC D) with β in adjacent monomer in APC12 ACS Paragon Plus Environment

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like trimer. Prior to Kuzminov and co-workers findings, Lijin and co-workers who also studied

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mutant Synechocystis sp. PCC 6803 found a fraction of small APC with an electronic transition

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at 660 nm, different from that at 663 nm.25 However, it is now established that the PBS of A.

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marina does not have these cofactors (β18 and LCM), which strength the hypothesis that the 655–

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665 nm and 667–675 nm transition may originate from two distinct exciton states. These

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excitons may be induced by the coupling between 84 (PC) and 84 (APC), and 84 (PC) and 84

249

(APC).

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The PBS in A. marina contains one APC trimer.14,

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Using site selective spectroscopy, an

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electronic transition of the APC trimmer was at 645 nm, which agrees with earlier finding by Hu

252

et al.26 The transition at 645 nm can be assigned to the exciton transition formed by APC trimer

253

in the PBS of A. marina. The electronic site peak at 670 nm suggests the presence of a terminal

254

emitter electronic transition. The peak 660 nm has been observed in the fluorescence of different

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PBS of Synechocystis PCC 6803 and T. vulcanus,1, 23 and it further supports that the structural

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shape of the PBS of A. marina allows the creation of the electronic transition necessary to

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transfer the phoexcitation to the reaction centre. Therefore, cyanobacteria can adapt the absence

258

of certain cofactor as 18 and LCM by turning the assembly shape of the PBS.

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Deactivation of the S1 electronic state by fluorescence. To better understand the coupling

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between cofactors in the excited state for efficient excitation energy transfer (EET), we first

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investigate the deactivation of S1 transition via fluorescence at 4K. Due to the local environment

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induced by the amorphous phase of the PBS at 4K, the absorbance shows an inhomogeneous

263

broader profile with rich local electronic transitions of distinct sub-protein complex (PC rod,

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APC core, LCM) in the PBS (also see Figure 1). First, the attention turned on the electronic

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transition that may occur in the PBS of T. vulcanus and A. marina when the high-energy cofactor 13 ACS Paragon Plus Environment


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β155 is preferentially excited around 615 nm. From Figure 3 (a), we observe that a first

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deactivation in PBS of T. vulcanus appears between 640–645 nm. This deactivation of the S1

268

transition between 640–645 nm tells us that a high-energy sub-compound exists because, due to

269

the excitation of the high-energy sub-compound, cofactors have been excited toward a

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deactivation between 640–645 nm. From literature, we learned that the PBS of T. vulcanus has

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PC620 that absorbs around 620 nm, while the PC612 may be located near the APC core as

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expected.10, 18 Since the electronic transition at 620 nm was already occupied by PC620, then we

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ruled out the hypothesis that the PC620 had been mainly excited at 615 nm due to its

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inhomogeneous broader absorption, and the obtained S1 deactivation around 640–645 nm was

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the product of its energy transfer from PC20 nm to PC612.


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Figure 3. Fluorescence after PC excitation of PBS of T. vulcanus (a, b) and PBS of A. marina (c,

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d). The absorbance of the respective PBS is shown in blue in the wavelength of interest. Features

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are labelled within the figures. The experiment was done at 4 K.

280

The energy transferred from PC620 initiated the activation mode in PC612. The next S1

281

transition appeared around 660 nm, which is the electronic transition involved the coupling of

282

18. 24, 27 The APC core composed of trimeric assembled disc had low energy electronic transition

283

at 652 nm as a result of excitonic splitting.28 To further transfer the EE to the terminal cofactor

284

electronic transition of LCM, the PBS needs additional electronic transition to cover the gap

285

between APC and the terminal emitter located around 675-680 nm. Therefore, with the present 15 ACS Paragon Plus Environment


Page 16 of 33

286

electronic transition observed on the spectrum (Figure 3a, 3b), the S1 transition at 660 nm can be

287

assigned to the coupling between β18 and β of a standard APC trimer in the core (β18 β). This may

288

allow the creation of an electronic transition where the EET was harvested at 660 nm, while the

289

low energy transition of the PBS of T. vulcanus at 680 nm was apparently LCM coupling with β

290

cofactor in a trimer like APC (scheme 2) LCMβ.

291 292

Scheme 2. Arrangement of APC containing monomer (Left) pdb ID 1B33 of APC disc

293

containing linker LC. (Right) proposed trimer like assembly of cofactor β18 encoded APC F and

294

LCM encoded apcE gene (APC E) form a monomer in a disc like – APC trimer.

295

At 630 nm, the electronic transition of β84 in PC was mainly excited.11 In any case the EET

296

arrived to the terminal emitter at 680 nm. Since αB encoded APCD was shown to be present in

297

the PBS of PBS of T. vulcanus1, and combined this information with the electronic transition

298

unveiled by Kuzminov and co-workers

299

terminal emitter induced by the coupling between LCM cofactor like-α and β in a trimer like-APC.

300

From the current result, we were able to monitor the EET from PC620 to APCE via PC612 and

301

APC F electronic transition at 645 and 660 nm, respectively (Figure 3a and 3b).

23

we can suggest the transition at 680 nm belong to the


Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


302

With regard to PBS of A. marina, excitation at 615, 620, 625 and 630 nm show an S1

303

deactivation electronic site at 645 nm (Figure 3c), which is the electronic transition of APC

304

cofactor. A shoulder appears between 635–638 nm. At this particular electronic transition, an

305

intermediate transient state was reported as the low energy state of PC in PBS of A. marina,22

306

which highlighted the result of Figure 2. Additional attention to this electronic site through

307

excitation of the 635 nm transitions reveals a clear change of the fluorescence profile. A plateau

308

appears before a deactivation electronic transition at 645 nm. This change on the phonon profile

309

compared to the profile induced by 630 nm excitation (Figure 3d) apparently originated in a

310

different activation mode, which is in line with the results of Figure 2. It is shown that there is

311

additional electronic transition that was hidden behind the inhomogeneous broader absorption of

312

cofactor in PC hexamer of PBS of A. marina.

313

After the excitation of different electronics transition in PC, the dissimilarity in fluorescence

314

profile between the PBS of T. vulcanus and A. marina was not sufficient to highlight the APC

315

contribution to the excitation energy transfer (EET) along the S1 transition mode deactivation at

316

660 nm. After excitation of APC in PBS of A. marina at 645 nm (Figure 4b), a double

317

vibrational mode appears at the phonon side band. This indicated that the vibrations are located

318

at the APC electronic site. The strength of the localized vibration of 40 cm-1 is reportedly

319

induced by the dimer cofactors α84 and β84 in APC.29 Besides, the one-phonon profile of the

320

fluorescence displays the deactivation of APC transition at 660 nm. However, this deactivation

321

of the S1 transition of APC is follow with the activation of the S1 transition of the cofactor at

322

660 nm, which is deactivated at 673 nm. The current transition is similar the transition marker

323

involved the coupling of the β18 cofactor at 660 nm.



Page 18 of 33

324

Alongside the 660 nm electronic transition, which is deactivated at 673 nm, the terminal

325

emitter of PBS of A. marina is 7 nm blue-shifted to that of PBS of T. vulcanus at 680 nm (Figure

326

4a). Regarding the 660 nm transition in the PBS of T. vulcanus, we can have apparent assurance

327

that the 660 nm electronic transition originated from APC emission via deactivation of S1 at 660

328

nm. There the coupling between β and β18 for APC trimer and trimer like-APC was expected to

329

be the acceptor of energy transfer from APC trimer, and the donor of the energy transfer to the

330

terminal emitter at 680 nm. Despite the bathochromic shift observed between the APC of PBS

331

of A. marina (645 nm) and PBS of T. vulcanus (652 nm), the two-antenna system displays an

332

apparent similitude to the APC emission via S1 deactivation electronic transition at 660 nm. The

333

differences appeared at the terminal emitter emission, which is located at 680 and 673 nm for

334

PBS of T. vulcanus and A. marina respectively.

335 336

Figure 4. (a) Non-line fluorescence of APC core in T. Vulcanus, (b) and APC trimer of A.

337

marina. The absorbance of the respective PBS is shown in read in the wavelength of interest.

338

Features are labelled within the figures.

339

We have assigned to the fluorescence the deactivation of the S1 transition in A. marina and

340

PBS of T. vulcanus. To shed more light on the comparison of the electronic transition 18 ACS Paragon Plus Environment

Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


341

deactivation between PBS of A. marina and T. vulcanus, we analyse the hole burning spectra

342

from the PBS of the two species, which is an absorption difference spectrum in steady state.

343 344

Deactivation of the S1 electronic state by absorption changes: Comparison of spectral hole

345

burning between the PBS of A. marina and T. vulcanus after excitation at 620 and 615 nm,

346

respectively, is shown in Figure 5. This ground state bleaching named spectral hole burning

347

(SHB) displays additional transition not observed on the fluorescence spectra. The PBS of T.

348

vulcanus displays a broad bleaching below 615 nm, with a minimum of the absorption change

349

trace close to 600 nm. The SHB of the PBS of A. marina shows a blue-shift bleaching at 615 nm.

350

Since there is no evidence of a cofactor absorbing resonantly at 615 nm, and in the absence of

351

exciton coupling between β155 and α84, the blue-shifted bleaching may perhaps be due to

352

formation of a pseudo-phonon side band as result of an excitation at 620 nm in PBS of A.

353

marina.

354

In T. vulcanus (Figure 5a), the observed electronic transitions at 624, 630, 637, 657-662, and

355

670-680 nm are due to the excitation energy transfer to the successive low electronic energy

356

sites. The 624 and 630 nm are known to be the resonance absorption of 84 and β84, repsectively,

357

while the 637 nm is due to the broadening of the transition at 635 nm, which is the high peak

358

absorption of the PC rod in PBS of T. vulcanus. The distortion of the bleaching trace between

359

640–645 nm is likely to be related to the activity of the cofactor present in PC612, which absorbs

360

near the APC core. However, instead of having bleaching of the APC core, we observe a slight

361

shoulder at the resonance absorption transition of APC core followed by a broad bleaching that

362

entirely covers the transition gap of APC F between 657–666 nm. The amplitude of the 657 and

363

637 nm is relatively of the same order of magnitude. This may indicate a very strong coupling 19 ACS Paragon Plus Environment


Page 20 of 33

364

between the 637 nm and 18 electronic transitions in PBS of T. vulcanus via APC core. Further

365

redshift bleaching appears between 670–680 nm, which is known as electronic transition of the

366

terminal emitter. We observe an important diminishing of the bleaching amplitude, which

367

indicates that a non-negligible fraction (80%) of the excitation energy transfer was trapped at the

368

(β-β18) transition.

369

The PBS of A. marina portrayed a completely different evolution of the EE flow from PC to

370

the terminal emitter (Figure 5b). After excitation at 620 nm, the energy flows at 630, 635, 645

371

and 673 nm. The first significant difference is the high amplitude bleached at 630 nm followed

372

by a localized doublet vibrational mode separated by 51 cm-1, a distinct bleaching of APC trimer

373

followed by a doublet vibrational mode of 40 cm-1 and finally the absence of 660 nm bleaching.

374

It is likely the trapping of excitation energy is located between 635 and 645 nm.

375

376 377

Figure 5. Comparison of spectral hole burning of a) PBS of T. vulcanus and b) PBS of A. marina.

378

The absorbance of the respective PBS is shown in red in the wavelength of interest. Features are

379

labelled in each graph.


Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


380

If the excitation of the PBS of T. vulcanus was turned to the β84 electronic transition at 630 nm

381

(Figure 6a), after a sharp phonon side band, the bleaching was observed at 640, 645, 660 and 675

382

nm. The bleaching of the 635‒637 nm electronic transition disappeared. A direct coupling

383

between 630 nm and 640 nm electronic transition was apparent. The shoulder at 645 nm

384

highlights the hypothesis that the electronic transition at 640 and 645 nm may be due to two

385

distinct cofactors in PC612. Furthermore, the relative amplitude of the (β-β18) at 660 was almost

386

equal to that of the excitation line at 630 nm, which further supports the idea of trapping of a

387

fraction of excitation energy at the (β-β18) electronic transition. The terminal emitter bleaching

388

was broader enough to cover the 680 nm transition, but it was centre at 675 nm, indicating that

389

perhaps an additional transition was present at the terminal emitter transition. Additional

390

bleaching was observed at 624 nm at the electronic transition of α84. Due to the excitonic

391

coupling between α84 and β84 in PC,22 excitation of α84 or β84 will induce the energy flow to its

392

homologue cofactor.

393

The excitation of PBS of A. marina at 634 nm induced a bleaching at 624, 635, 640 and 673

394

nm (Figure 6b). The 624 nm is distinctive to the pseud-phonon side band, which was signalling

395

with a slight shoulder near the excitation transition. The possible explanation of this blue-shift

396

bleaching can be attributed to an exciton coupling in which the high-energy exciton funnels the

397

excitation energy to the low energy one and vice versa. The significant discrepancy between A.

398

marina and PBS of T. vulcanus was the low amount of excitation energy that arrived at the PBS

399

of A. marina terminal emitter and the absence of the (ββ18) bleaching.



Page 22 of 33

400 401

Figure 6. Spectral hole burning of a) PBS of T. vulcanus after excitation at 630 nm and b) PBP

402

rod A. marina after excitation at 634 nm. The absorbance of the respective PBS is shown in red

403

in the wavelength of interest. Features are labelled in each graph.

404

Further insight into the electronic structure of the intact T. vulcanus and PBS of A. marina is

405

gained via excitation of the APC sub-compound (Figure 7). One of the main characteristics of

406

the APC trimer was the difference of its electronic structure compared to that of PC trimer. The

407

exciton strength was suggested to be higher in APC.22,

408

excitation of APC core absorption gap in PBS of T. vulcanus at 652 nm was characteristic, with a

409

broad blue-shifted bleaching in the entire PC rod, as will be observed between two exciton states

410

(Figure 7a). The excitation of the low energy exciton induces an energy flow to the high-energy

411

state this and vice versa. As expected after the excitation of APC T. vulcanus takes place, a more

412

pronounced bleaching was observed at 657 nm ((β-β18) APC F) followed by the bleaching of the

413

terminal emitter. The relative amplitude of the bleaching at 658 nm compared to the other

414

bleaching (PC rod, APC at 652 nm, terminal emitter) suggested that most of the energy was

415

trapped at 660 nm. Another feature was the broad bleaching of the terminal emitter APC E

416

between 670‒688 nm. A contrasting observation was obtained in the case of PBS of A. marina

29-30

The obtained signal trace after


Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


417

(Figure 7b). Almost no bleaching of the β-β18 (660 nm transition) and the terminal emitter was

418

observed. Besides the 635 nm blue-shifted bleaches, there was no clear evidence of broad

419

delocalize blue-shifted bleaching on the entirely PC hexamer. For the case of PBS of T.

420

vulcanus, the end of the broad blue-shifted bleaching is well demarked on the signal trace at 690

421

nm.

422

423 424

Figure 7. Comparison of spectral hole burning of a) Tv PBS and b) Am PBS. The absorbance of

425

the respective PBS is shown in red in the wavelength of interest. Features are labelled in each

426

graph.

427

Since the PBS of A. marina and T. vulcanus had the 670 nm transition due to the coupling

428

between APC E cofactor like α and β in a trimer-like APC (in T. vulcanus), the excitation was

429

done at the 670 nm transition instead of 680 nm for comparison purposes (Figure 8). Indeed,

430

only the PBS of T. vulcanus showed the excitement. The result displayed in Figure 8 gives

431

additional information on the coupling between APC (APC trimer for PBS of A. marina and APC

432

core at 652 nm for PBS of T. vulcanus) and the terminal emitter electronic transition. In Figure

433

8a, the evolution of absorption trace shows a weak bleaching of the APC F coupling between

434

657‒666 nm and a more significant bleaching of the APC core at 652 nm. The weak bleaching 23 ACS Paragon Plus Environment


Page 24 of 33

435

around 657– 666 nm highlights the hypothesis of a trapping site of β-β18 as an upper state of the

436

terminal emitter in PBS of T. vulcanus. Since the excitation was deposited on the terminal

437

emitter, there was no need for the (β-β18) APC F to control the energy flow to APC E electronic

438

transition β-LCM. The slight bleaching can now be interpreted as a switching off of the trapping

439

that monitors the flow of the energy from the upper energy state of the antenna system (PC,

440

APC) to the low energy state β-LCM, the terminal emitter. It further looks like the β-LCM, β-β18

441

and APC core (652 nm) are strongly coupled. If this were not the case, the blue-shift bleaching at

442

652 nm after excitation of β-LCM transition at 670 nm would not appear. A clear difference

443

appeared when the terminal emitter of PBS of A. marina was excited (Figure 8b). Indeed, the

444

similar bleaching as of β-β18 electronic transition in T. vulcanus was observed at 663 nm. The

445

obtained signal, likely a weak bleaching of APC trimer at 645 nm, was observed. We were not

446

able to obtain a better resolution. This latter aligns with the previous excitation done in APC at

447

645 nm (Figure 7b), in which no apparent evidence of the terminal emitter bleach was observed.

448 449

Figure 8. Hole burning spectrum after excitation of the terminal emitter of a) PBS of T. vulcanus

450

and, b) PBS of A. marina. The absorbance of the respective PBS is shown in red in the

451

wavelength of interest. Features are labelled in each graph. 24 ACS Paragon Plus Environment

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452

New perspectives. The excited electronic delocalized over photosynthetic pigment has been

453

useful for describing the mechanism of EET in a light harvesting system. This delocalization of

454

the excited electronic state known as exciton originated from columbic interaction between

455

pigments that are suggested to be identical in the system.31-32 Using a dimer pigment approach of

456

exciton mechanism, it was proposed that the importance of the splitting and the oscillator

457

amplitude strength are related to the orientation of the coupled dipole moments.33 The well-

458

organized pigment protein structure in PBS favours flows of the excitation energy from the upper

459

energy to the lower one. In the case of strong columbic interaction between pigments in two

460

different sub-compounds, where one was at the high electronic energy level and the second was

461

at the low electronic energy level, we proposed that when excited at the high energy level, the

462

excitation energy flows to the low energy. This will be vice versa, meaning full, when the low

463

electron energy was excited. It was expected to induce bleaching of the high energy level as well

464

as the low energy level. This approach can be extended to 3 sub-components, which are strongly

465

coupled in an electrostatic manner. However, because of the organization of pigment-pigments

466

and pigment-proteins to funnel the energy transfer from the high electronic to the low electronic

467

energy level, increased bleaching may be expected when the high electronic energy level was

468

excited compared to the low electronic energy level. We do not pretend that this was a full

469

mechanism that was hidden in this coupling between sub-compounds, but it is still an approach

470

that leads to better understanding of the bleaching we obtained when the excitation was turned

471

from high or for low electronic energy vice versa (scheme 3). Figure 7a and 8a are examples,

472

allowing us to suggest that APC Core, trimer like-APC containing a monomer of β18 (APC F)

473

and LCM (APC E) are strongly coupling according to the aforementioned mechanism. A possible

474

Hamiltonian might be:



475 476

H = H[coreAPC] + H[(ββ18)APC F] + H[(βL

CM

APC F+(βLCM) APC E ] +

)APC E]

Hcoupling[(ββ18) APCF + (βL

CM

Page 26 of 33

+ Hcoupling[coreAPC, [(ββ18) APC F] + Hcoupling[coreAPC, [(ββ18)

)APC E]

477 478

Scheme 3. Illustration of exciton effects on the energy level in a possible degenerate trimer

479

coupling of the sub-compound: Ee and Eg represent the ground and excited electronic states. D

480

represents the dispersion function that contains the non-bounded interactions; hu, hi, and hl

481

represent the upper, intermediate and lower electronic states, respectively. In the right side of the

482

graphic, the black, red and blue curves represent a schematic of respective amplitude from APC

483

core, (β-β18) APC F and (β-LCM) APC E. The orientation of the dipole moments is expected to

484

influence the splitting amplitude. Modified from Davydov 33.

485

If the excited energy was deposited on the electronic transition of (β-LCM) APC E, the

486

bleaching of the (β-β18) APC F electronic transition was considerably reduced. However, if this

487

excited energy with the same fluence was deposited at the high electronic energy level as core

488

APC (652 nm) (Figure 7a) or a higher electronic energy level (PC) (Figures 6a and 5a), the

489

amplitude of the bleaching of the electronic transition (β-β18) APC F increased. The increased 26 ACS Paragon Plus Environment

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490

bleaching amplitude of the electronic transition of the (β-β18) APC F was even more than the

491

bleaching of the excited line gap at high electronic energy level (Figure 5a). A possible

492

explanation of this change in the bleaching in the (β-β18) APC F electronic transition was that a

493

considerable fraction of the excited energy was trapped at the (β-β18) APC F electronic site.

494

According to the bleaching amplitude (see Figures 5a, 6a, 7a and 8a), 80% of the excitation

495

energy was trapped at the (β-β18) APC F electronic site. This trapping of the excited state

496

population at the (β-β18) APC F transition was switched off when the (β-LCM) APC E electronic

497

transition was excited instead. This suggests that APC F at 660 nm may regulate the excitation

498

energy that flows from the high electronic energy level to the terminal emitter APC E in PBS of

499

T. vulcanus. This mechanism, which we named APC F switching, seems to be absent in PBS of

500

A. marina. The reason remains unknown, which call to more investigation of the PBS of A.

501

marina.

502

However, the 673 nm electronic transition was found in both PBS species (A. marina and T.

503

vulcanus). Although (β-LCM) APC E is only present in T. vulcanus, the presence of this energy

504

transition in PBS of A. marina may suggest that there is structural adaptation of APC and PC

505

cofactor assembly, creating the low electronic energy levels in the A. marina PBS. However, the

506

apparent absence of the lowest electronic transition of (β-LCM) APC E, the 680 nm in PBS of A.

507

marina, may have an unknown structural relevance. This may also suggest an additional effort

508

on the PBS of A. marina purification to avoid the loss of a fraction of terminal linker.

509

Furthermore, the PBS of both T. vulcanus and A. marina exhibits electronic transition at 660

510

nm, which was characteristic of (β-β18) APC F electronic transition (Figure 4). Despite 7 nm

511

blue-shifted spectral position of APC in PBS of A. marina compared to PBS of T. vulcanus, A.

512

marina conserved the electronic transition at 660 nm. Presumably, the structural organization of



Page 28 of 33

513

the PBS of A. marina, which turned the blue-shifted electronic transition of APC trimer at 645

514

nm, has an effect on the non-bounded interaction of the successive phycobilin cofactor. The

515

Figure 8b had showed no apparent bleaching of APC trimer in PBS of A. marina, which

516

highlighted our hypothesis about the weak coupling between APC trimer and the terminal

517

emitter in PBS of A. marina compared to PBS of T. vulcanus.

518

Additionally, the APC core excitation 7a (PBS of T. vulcanus) compared to the excitation of

519

APC trimer 7b (PBS of A. marina) had showed an apparent blue-shift bleaching in all PC

520

electronic transitions of PBS of T. vulcanus, while the delocalized blue-shifted hole in PC of

521

PBS of A. marina appeared at the level of noise. Increasing the concentration of the PBS of A.

522

marina, or the fluence, did not bring a clearly distinguished blue-shifted bleaching in PC when

523

excited by the APC trimer. This observation aligned with the excitation of the terminal emitter in

524

Figure 8b. Because of this observation, it appeared that the coupling strength between APC

525

(APC trimer for PBS of A. marina and APC core for PBS of T. vulcanus) and PC in PBS of T.

526

vulcanus was more important than in PBS of A. marina. This hypothesis can be extended to the

527

coupling between APC, (β-β18) APC F and (β-LCM) APC E (the terminal emitter), and he

528

terminal emitter of A. marina PBS.

529

CONCLUSION

530

With the results above, we have proved beyond reasonable our hypothesis that cryo-

531

spectroscopy of intact PBS reveal intrinsic EET in real-time, closer to how it happens in the

532

natural light-harvesting antenna systems PBS of A. marina and T. vulcanus. The study resolved

533

most of EET mechanism obscured by noise coming from thermal coupling of PBS components

534

at room temperature. Further, the overall EET processes, previously not observed in extract, have

535

been clarified through the use of intact PBS. For instance, we now know that there is a weak

536

similarity of their electronic transition states the PBS’s of A. marina and T. vulcanus. Aside from 28 ACS Paragon Plus Environment

Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


537

the electronic transition that appears at 660 nm, a complete blue-shift absorbance spectra from

538

the PBS of A. marina and T. vulcanus has been observed and compared. This blue-shift

539

absorption affects the coupling interaction that generates the energy flow in the PBS of A.

540

marina. Further, we managed to find the 635 nm in the PC electronic transition in PBS of A.

541

marina. Regarding the terminal emitter transition, the PBS of T. vulcanus had more low

542

electronic energy transitions than PBS of A. marina. This difference appeared at 680 nm that was

543

observed only in the PBS of T. vulcanus. Additionally, the PBS of T. vulcanus regulated the

544

excitation energy that arrived at the terminal emitter (β-LCM) APC E electronic site by trapping

545

80% at the (β-β18) APC F electronic transition. β155 in high electronic energy level in PC seems

546

to be strongly coupled in PBS of T. vulcanus as a consequence of the PC geometry. All these

547

information have never been so clear in spectroscopic studies of PBS extracts at room

548

temperature.

549 550

AUTHOR INFORMATION

551

Corresponding Author

552

*Martin Mkandawire, Tel. +1-902-563-1430; Fax. +1-902-563-1360; Email:

553

[email protected].

554

Funding Sources

555

This work has been made possible with financial support from formally Enterprise Cape Breton

556

Corporation (ECBC) to the Industrial Research Chair for Mine Water Management at Cape

557

Breton University, the US-Israel Bi-National Science Foundation (2009406), the Israel Science

558

Foundation founded by the Israel Academy of Sciences and Humanities (1576/12).



Page 30 of 33

559

ACKNOWLEDGMENT

560

Authors are also thankful for kind support from Prof. David W. McCamant of Chemistry

561

Department at University of Rochester. They are also grateful to Ms. Julie Small of Dalhousie

562

University (Truro Campus) for proofreading and considerably improving of the language of the

563

first draft of the manuscript.

564

ABBREVIATIONS

565

PBS = phycobilisomes; PC = phycocynine; APC = Allophycocynine; LCM= XX; EET=

566

excitation energy transfer;

567

REFERENCES

568

1.

David, L.; Prado, M.; Arteni, A. A.; Elmlund, D. A.; Blankenship, R. E.; Adir, N.,

569

Structural studies show energy transfer within stabilized phycobilisomes independent of

570

the mode of rod–core assembly. Biochimica et Biophysica Acta (BBA) - Bioenergetics

571

2014, 1837 (3), 385-395.

572

2.

Tiwari, V.; Peters, W. K.; Jonas, D. M., Electronic resonance with anticorrelated pigment

573

vibrations drives photosynthetic energy transfer outside the adiabatic framework.

574

Proceedings of the National Academy of Sciences of the United States of America 2013,

575

110 (4), 1203-1208.

576

3.

Liu, H.; Zhang, H.; Niedzwiedzki, D. M.; Prado, M.; He, G.; Gross, M. L.; Blankenship, R.

577

E., Phycobilisomes Supply Excitations to Both Photosystems in a Megacomplex in

578

Cyanobacteria. Science 2013, 342 (6162), 1104-1107.

579

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Kreisbeck, C.; Kramer, T.; Aspuru-Guzik, A., Scalable High-Performance Algorithm for

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the Simulation of Exciton Dynamics. Application to the Light-Harvesting Complex II in

581

the Presence of Resonant Vibrational Modes. Journal of Chemical Theory and

582

Computation 2014, 10 (9), 4045-4054.

583

5.

Romero, E.; Augulis, R.; Novoderezhkin, V. I.; Ferretti, M.; Thieme, J.; Zigmantas, D.;

584

van Grondelle, R., Quantum coherence in photosynthesis for efficient solar-energy

585

conversion. Nature Physics 2014, 10 (9), 676-682. 30 ACS Paragon Plus Environment

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Frontiers in Plant Science 2014, 5, 7.

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