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Spectral Hole Burning in Cyanobacterial Photosystem I with P700 in Oxidized and Neutral States Nicoleta Herascu, Mark S. Hunter, Mehdi Najafi, T. Wade Johnson, Petra Fromme, Golia Shafiei, and Valter Zazubovich J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b07803 • Publication Date (Web): 23 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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The Journal of Physical Chemistry

Spectral Hole Burning in Cyanobacterial Photosystem I with P700 in Oxidized and Neutral States Nicoleta Herascu1, Mark S. Hunter2,+, Mehdi Najafi1, T. Wade Johnson3, Petra Fromme2, Golia Shafiei1and Valter Zazubovich1,* 1

Department of Physics, Concordia University, 7141 Sherbrooke str. West, Montreal, H4B 1R4, Quebec, Canada; 2 Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA; 3 Department of Chemistry, Susquehanna University, Selinsgrove, PA, USA.

* Corresponding author; E-mail: [email protected]. Phone (514) 848-24-24 ext 5050. +

Current address: Lawrence Livermore National Laboratory, Livermore, California 94550,

USA

Abstract: We explored the rich satellite hole structures emerging as a result of spectral hole burning in cyanobacterial Photosystem I (PSI) and demonstrated that hole burning properties of PSI, particularly at high resolution, are strongly affected by the oxidation state of the primary donor P700 as P700+ effectively quenches the excitations of the lowest-energy antenna states responsible for fluorescence. Obtaining better control of this variable will be crucial for highresolution ensemble experiments on protein energy landscapes in PSI. The separate NPHB signatures of various red antenna states were obtained, allowing for additional constraints on excitonic structure-based calculations. Preliminary evidence is presented for an additional red state of PSI of T. Elongatus peaked at 712.6 nm, distinct from previously reported C708 and C715 states and possibly involving chlorophyll B15. Excitation at wavelengths as long as 800 nm results in charge separation at cryogenic temperatures in PSI also in Synechocystis sp. PCC 6803. Both the “P700+ minus P700” holes and non-photochemical spectral holes were subjected to thermocycling. The distribution of barriers manifesting in recovery of the “P700+ minus P700” 1

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signature contains two components in sample-dependent proportions, likely reflecting the percentages of FA and FB clusters being successfully pre-reduced before the optical experiment. The barrier distribution for the recovery of the lower-energy non-photochemical spectral holes resembles those observed for other pigment-protein complexes, suggesting similar structural elements are responsible for NPHB. Higher-energy components exhibit evidence of “domino effects” such as shifts of certain bands persisting past the lower-energy hole recovery. Thus, conformational changes triggered by excitation of one pigment likely can affect multiple pigments in this tightly-packed system.

1. Introduction. Photosystem I (PSI) is one of the two major protein complexes involved in oxygenic photosynthesis. Its main function is to catalyze light-driven electron transport across the thylakoid membrane. The detailed 3D structure of PSI is available, both for cyanobacteria and plants trimeric

1-9

1-3,8

. PSI is present in monomeric form in plants, while in cyanobacteria it is usually

. In cyanobacterial PSI membrane-intrinsic peripheral antennae are not present,

except in the case of iron stress

10,11

. The main core subunits PsaA and PsaB are surrounded by

several small transmembrane proteins that stabilize the complex and mediate trimerization of cyanobacterial PSI

12

. In all organisms PSI is a joint reaction center and core antenna system,

thereby the reaction center of PSI (PSI-RC; Figure 1A) is an integral part of the PSI core. Respective cofactors of the electron transport chain are coordinated by the PsaA, PsaB and PsaC protein subunits, with PsaA and PsaB also coordinating most of the core antenna chlorphylls and carotenoids. The primary electron donor in PSI (P700) is supposed to be the “special pair”, a chlorophyll a / chlorophyll a’ heterodimer that is located close to the lumenal side of the membrane, but there is also spectroscopic evidence that charge separation can initiate at eC2/eC3 pair in the electron transport chain of PSI

13

. The efficient excitation energy transfer (EET) 2


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within the antenna network and to the RC is followed by charge separation between P700 and primary acceptor, A0 (chlorophyll a, Chl a; green in Figure 1A), which very likely involves the accessory Chl a. The electron is further transferred to the intermediate electron acceptor A1 (phylloquinone; yellow in Figure 1A) 9, then to the Fe4S4 cluster FX and to the terminal electron acceptors FA and FB (also Fe4S4 clusters). A variety of experiments suggest that P700 absorption is peaked at around 700 nm

14-17

. It was also noticed that absorption, electron paramagnetic

resonance (EPR) and magnetic circular dichroism (MCD) signals in PSI extend beyond 800 nm18-21 and that excitation at these wavelengths could result in charge separation20,21 in some plants and cyanobacteria. Similar long-wavelength charge separation was observed also for PSII and attributed to direct excitation of the low-laying charge-transfer (CT) states of the RC 18,19,22,23

.

In both plants

24,25

and cyanobacteria

17,26-30

several antenna chlorophylls absorb at

wavelengths significantly longer than 700 nm. These chlorophylls are known as “red chlorophylls” and respective bands - as “red antenna states”, and their number and peak absorption and emission wavelengths vary between the species. The red shift of these states is attributed to the combination of the red shifts of the site energies (transition energies in the absence of inter-pigment coupling) of some pigments due to interactions with protein environment and strong electrostatic interactions within the groups of closely-spaced pigments leading to excitonic splitting. There are also indications that some of them may possess charge transfer character

28,27,29

. At physiological temperatures these states can transfer energy uphill to

the P700, and at low temperatures they may act as competing energy traps

17,24-39

. The likely

purpose of these chlorophylls is to extend the wavelength range of harvested light. The lowestenergy states of PSI from several cyanobacteria were identified and competing assignments of

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these states to particular groups of the strongly coupled Chls were proposed

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1,2,29,30,32-40

. The

groups of chlorophylls traditionally considered as the origin of the “red states” are shown in Figure 1B. B37-B38 and A38-A39 are located on the stromal side of the complex, while B31B32-B33 and B7-A32 are located on the lumenal side (see Figure 1). Synechocystis sp. PCC 6803 exhibits two pools of red chlorophylls, C706 and C714 least three red antenna states, C708, C719

17,29

and C715

29

26,27,39

, while T. elongatus has at

labeled according to their peak

absorption wavelengths. The lowest-energy states of T. elongatus and Synechocyctis sp. PCC 6803 exhibit similar properties (very strong electron-phonon coupling, large pressure-induced shift and strong Stark effect

28,27,29

) which suggests that these states likely originate from a

chlorophyll cluster whose structure is preserved across species and that these states possess significant CT character. However, certainty concerning the origins of each of the red states has not yet been reached. Theories with different levels of complexity (dipole-dipole approximation is too crude for inter-pigment distances less than one nanometer

32-34,40

) assign the strongest

inter-pigment couplings to different groups of pigments. There are more Chl a clusters with strong inter-pigment couplings (|Vab| >100 cm-1) than there are “red antenna states”

32-34,40

.

Moreover, there is no agreement concerning the exact site energies (transition energies in the absence of inter-pigment interactions) of the chlorophylls in PSI 33-35,40. Therefore, assignment of the red states requires additional comparisons between structural and spectroscopic data. Most of these comparisons were so far performed for T. elongatus, for which the X-ray structure has been available for years

1-3

. Recently this structure was refined using DFT

41

. The structure of PSI

from Synechocystis sp. PCC 6803 was reported very recently, and is based on a genetically modified PSI monomer 42.

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Most reports agree on the lowest-energy state (C719 in T. elongatus and C714 in Synechocystis sp. PCC 6803) being attributed to the B7-A32 dimer

29,30,37,40

or to the more

extended B6-B7-A31-A32 pigment cluster. There is also evidence that one of the red states is likely due to the B31-B32-B33 trimer

38,40

. The amplitude of the longest-wavelength absorption

band is reduced upon the monomerization of PSI

17,26

, and therefore respective chlorophylls are

likely located close to the symmetry axis of the PSI trimer. Schlodder et al. have shown that the fluorescence of the red-most antenna state in cyanobacterial PSI (i.e., C719/F740 in T. elongatus) can be suppressed due to the effective downhill EET to the oxidized P700 (P700+) 31, indicating that this group of antenna chlorophylls must be located also relatively close to the special pair (and excluding, for instance, the B31-B32-B33 trimer as a likely candidate for C719/F740; one may also note that B31-B32-B33 trimer is not present in Synechocystis sp. PCC 6803

42

). The local environment of B7-A32 is different in T. elongatus and Synechocystis sp.

PCC 6803, although it is not clear to what extent this is merely the result of the different preparation procedure of the Synechocystis PSI 42. In T. elongatus the peak of the emission band shifts by almost 10 nm depending on the redox state of P700

31

. When P700 is oxidized, the

lowest C719/F740 state is quenched due to EET to P700+, its lifetime is shortened and the emission and the NPHB spectra are likely dominated by the second-lowest C715 state, emitting at 732 nm. Absorption of P700+ extends to lower energies than that of the C719/F740 state and downhill EET from this state to P700+ is rather likely. Fluorescence of the other red states appears less sensitive to the redox state of P700 and their structural origins are less clear 31. The C708 state of T. elongatus exhibits much lower electron-phonon coupling than C719 and C715 28

, and, as indicated by the Single Photosynthetic Complex Spectroscopy (SPCS) experiments,

emits around 710-712 nm 30,36-38.

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Figure 1. Frame A: Side view on the structural arrangement of the cofactors in the reaction center of PSI (PDB: 1JB01). The arrows indicate the course of the charge separation. Blue: special pair, P700; Red: accessory chlorophylls Chlacc; Green: Chl A0 ; Yellow: phylloquinone (PhQ), A1; the terminal electron acceptors are iron-sulfur clusters FX, FA and FB (red/yellow). Frame B: View of the PSI core monomer from the stromal side of the membrane with the most likely candidates for the “red antenna states” 2,34 being shown with bolder lines. Color coding of the RC cofactors is the same as in frame A. The B7-A32 dimer is highlighted in purple, other suspected “red state” clusters are highlighted in red. The rest of the antenna chlorophylls are green. The C3 axis of symmetry of the PSI core trimer is located above the depicted PSI monomer, not far from the B7-A32 and B37-B38 dimers, and is perpendicular to the plane of the page (triangle).

Both spectral hole burning and SPCS were employed in recent studies of the “red antenna states”26,27,29,30,32-39,43-45. SPCS is particularly suitable for observing the shifts of transition energies of various states. Brecht and co-workers proposed utilizing the dynamics of these shifts as an argument influencing the assignments of the “red states” to specific chlorophyll clusters 38. These shifts may be spontaneous or light-induced, in the latter case representing the elementary

6


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acts of non-photochemical spectral hole burning (NPHB). In NPHB optical excitation triggers a small structural change in the environment of the pigment molecule, in turn resulting in the shift of the pigment’s transition energy

46,47

. The pigment molecule itself does not experience any

chemical reaction and the shifted absorption of the burnt molecules forms the “anti-hole” that is usually blue-shifted with respect to the original hole. Thus, the NPHB spectral holes are qualitatively different from the features due to charge separation, even though both types of holes could persist for days at cryogenic temperatures.

The dependence of the rates of the

single molecule line jumps (spectral diffusion) on the composition of the environment surrounding the protein (regular versus deuterated buffer, glycerol, PVA, etc,) suggests that spectral dynamics observed in SPCS experiments on PSI involve proton tunneling 43-45. Another very interesting SPCS observation on PSI involves switching between alternating energy transfer pathways as a result of light-induced conformational changes (i.e. NPHB) in the protein 43. (The NPHB shifts of the EET donor or acceptor spectra may result in significant changes in the spectral overlaps.) These SPCS results served as the initial motivation for research reported in this manuscript. Over the last several years we performed non-photochemical hole-burning (NPHB) experiments on several other pigment-protein complexes involved in photosynthesis

48-51

, and

developed novel approaches to modeling NPHB and hole recovery yielding the distributions of barriers on the protein energy landscapes. However, with the exception of LH2

48

our NPHB

experiments were performed on complexes not yet explored by SPCS. Therefore, comparing spectral dynamics of PSI observed using NPHB and SPCS was an obvious step. Exploring the protein dynamics in PSI by means of high-resolution NPHB, however, is not an easy task. The lowest-energy state of the PSI exhibits very strong electron-phonon coupling and therefore it is

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impossible to burn more than ~10-15% zero-phonon holes (ZPH) in it 29. If the emission of the C719 state is indeed peaked at 741 nm 31, the electron-phonon coupling for the C719 state might be even stronger than previously estimated 29. The zero-phonon holes (ZPH) observed so far at around 720 nm

29

might have partially or even fully originated from the C715 state instead. The

hole growth kinetics

48-51

(HGK) and hole recovery experiments on the C719 band would suffer

from low dynamic range even under otherwise ideal conditions. The lifetimes of this and other red states must be affected by the EET to the lowest-energy antenna state and/or P700 (including CT contributions

18

) and P700+, and the EET rates and rate distributions are unknown.

Determining these distributions would be of interest as well, as this could provide further constraints for assigning red states to particular Chl a clusters. An interesting way to explore the “red states”, and an alternative to the higher-resolution NPHB for the purpose of determining relevant distributions of barriers on the protein energy landscapes, is to investigate the behavior of the satellite hole structures emerging upon illumination of PSI at various wavelengths upon subsequent thermocycling

50-54

. This satellite hole structure may include the upper excitonic

components of the lowest-energy states, and possibly some features due to the electrochromic shifts of the bands belonging to pigments in the vicinity of either the P700, or of the pigments responsible for the red states, given that the latter possess significant CT character 26,27,29 .

2. Experimental. We performed low-temperature (5 K) NPHB and thermocycling experiments on PSI from T. elongatus and Synechocystis sp. PCC 6803. The T. elongatus sample was produced by first dissolving the ultra-pure crystals of PSI employed in X-ray diffraction experiments

55

in a small

amount of buffer containing 5 mM MES, pH=6.4, 100 mM MgCl2 and 0.02% β-

8


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dodecylmaltoside. Synechocystis samples were never crystallized and were produced employing a more conventional protocol

26

. A Synechocystis menB mutant, which contains plastoquinone

instead of the phylloquinone 56, was also explored. Highly-concentrated samples (~10-4 M Chl a, OD~10 at the Qy peak) were diluted to achieve peak Qy OD of about 0.6 near 680 nm for 10 mm optical path. This corresponds to Chl a concentration of ~10-5 M and RC concentration of ~10-7 M. Three types of sample preparations were explored. In the case of samples of the type R (Reduced), PSI concentrate was diluted with a buffer containing 100 mM CAPS (pH 9.5-10), 10 mM MgCl2, and 0.02% β-dodecylmaltoside detergent 31. They were further mixed with glycerol (1:2) serving as cryo-protectant and ensuring formation of transparent low-scattering glass at low temperatures. As glycerol is known to damage some pigment-protein complexes, care has been taken to minimize the time that glycerol-containing samples spent at room temperature. This prevented damage to the sample (various optical spectra did not change for samples frozen and thawed several times), but certain homogenization of spectral properties of individual complexes 45,57

has likely occurred. Immediately prior to the experiment sodium dithionite was added (final

concentration of 20 mM) to ensure pre-reduction of the iron-sulfur clusters of PSI 31. This prereduction of the terminal Fe4S4 clusters to FA- and FB- blocks electron transfer from P700 all the way to FA and FB. As a result, only the charge separated states P700+ A0- or P700+ FX- are allowed to form upon illumination, which recombine to P700 A0 or P700 FX within 170 µs and 5100 ms, respectively

16,30

, i.e. there should be no persistent “P700+ minus P700” features in the

spectra. Samples O (Oxidized) were dissolved in 20 mM tricine, pH=7.5, 25 mM MgCl2, 0.02% β-dodecylmaltoside buffer 31 and cooled down under intense illumination (using a Thorlabs lamp and/or a LED-based flashlight). This resulted in the samples with P700 oxidized to P700+ before the beginning of the optical experiment, and (almost) no permanent P700+ minus P700 features

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in the difference spectra. Samples PR (Partially Reduced) were the same as samples R, except one third the amount of dithionite was added. PR samples were cooled down in complete darkness. As a result, the samples turned out with iron-sulfur clusters being preferentially, but not fully pre-reduced, and easy formation of persistent (at low temperature) spectral features due to P700 oxidation was possible. The NPHB experiments were performed in either fluorescence excitation mode at high resolution or (mostly) in transmission / absorption mode at lower resolution. The absorption spectra were measured with a Varian Cary 5000 spectrophotometer at 0.25 nm resolution while the fluorescence excitation spectra were recorded using a Hamamatsu PMT / photon counter module through 750 nm long-pass interference filter (Omega). The tunable Spectra Physics model 3900 Titanium-Sapphire laser (bandwidth ~30 GHz), the frequency-doubled CW Nd:YAG laser (Spectra Physics Millennia, 532 nm) and the tunable Sirah Matisse dye laser (LDS698 for 675-730 nm or DCM for 650-690 nm; bandwidth < 1 MHz) were employed. The latter laser was used for both hole burning and reading of the spectra (at low intensity) in the fluorescence excitation mode. Care was taken to minimize the amount of light reaching the sample prior to the optical measurements, especially those involving exploration of the P700+ formation (see above). The spectrometer and the laser system were located in different rooms connected by the optical fiber. The spectrometer room was kept as dark as possible, several nW/cm2, all wavelengths combined. The sample compartment of the spectrophotometer was further carefully isolated from the external stray light, with the only remaining aperture being covered with long-pass interference filters (Omega). The sample was placed in a liquid helium cryostat by the Ukrainian Academy of Sciences

58

. Thermocycling involves performing hole

burning at low temperature (e.g. 5 K), temporarily elevating the temperature, measuring the hole

10


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spectrum again at low (burn) temperature, and repeating this sequence with increasing maximal cycle temperature until the hole recovers by burnt systems crossing the barriers on their way back on the energy landscape.

3. Results: 3.1. Absorption and emission spectra of the samples with P700 in various states of oxidation are shown in Figure 2. The shapes of the absorption spectra for both T. elongatus (black, a) and Synechocystis sp. PCC 6803 (blue, b) in Figure 2A did not depend noticeably on the oxidation state of P700. The relative magnitudes of the red states and the sharpness of the peaks and shoulders, particularly in the case of T. elongatus, indicate that our samples are trimeric and of high quality. In T. elongatus (black) employing reductant results in a significant shift of the emission band (Figure 2B). For mostly reduced sample a weak shoulder at about 730 nm in addition to the main band at 741 nm is visible. The effects of the oxidation state of P700 on the shape of Synechocystis sp. PCC 6803 emission spectra (blue) are not as large. We noticed, however, that the fluorescence of the oxidized samples was significantly less intense (for samples of comparable optical density) than for the reduced samples, confirming quenching of red-states fluorescence by P700+ also for Synechocystis.

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Figure 2. Frame A: Absorption spectra of trimeric PSI from T. elongatus (black) (a) and Synechocystis sp. PCC 6803(blue) (b). Frame B: PSI fluorescence spectra excited at 651 nm for mostly reduced T. elongatus (a-R) (black), oxidized T. elongatus (a-O) (black dotted), reduced Synechocystis (b-R) (blue) and oxidized Synechocystis (b-O) (blue dotted). 3.2. Persistent P700+ Minus P700 Signature. For both species illumination of the partially reduced (PR) samples yields a strong persistent P700+ minus P700 signature. 3.2.1 T. elongatus. The most prominent bleach is at 703 nm, there also is a prominent positive feature at 691 nm, negative doublet at 684 nm and 687 nm, as well as a number of smaller and narrower features (Figures 3 A and B). Spectra are shifted vertically with respect to each other for clarity. Based on both peak magnitude and integral intensity of the 703 nm bleach, we estimate that in about 10% of the complexes in our T. elongatus PR sample P700 could still be photo-oxidized and terminal iron-sulfur clusters could be correspondingly persistently reduced at low temperatures. Note that complexes which have either oxidized P700 or both iron-sulfur clusters successfully pre-reduced from the beginning would not contribute to the persistent P700+ minus P700 signature. The former are already oxidized, and in the latter recombination occurs 12


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within milliseconds


17,31,59

. The persistent P700+ minus P700 signature originates only from the

complexes that originally had P700 in the reduced state as well as had the terminal iron-sulfur clusters available. Judging by the fluorescence peaked at 741 nm (Figure 2B), the rest of the complexes in this sample were in the state with successfully pre-reduced FA- and FB- iron-sulfur clusters. The shape of our P700+ minus P700 signature closely matches those presented in earlier works, see e.g. ref 21. This signature difference spectrum could be produced by exceedingly weak as well as by fairly red-shifted illumination. Green curve in Figure 3A is the difference between the second and the first absorption spectra measured with a Cary 5000 spectrometer after cooling the sample down to 5 K in the dark. Blue curve is the difference spectrum produced by one minute exposure to the dimly lit room (one computer monitor facing away from the spectrophotometer; several nW/cm2 for all wavelengths combined; no interference filters). Deliberate illumination of the sample with the laser produced P700+ minus P700 spectrum of larger amplitude. Black curve in Frame A is a result of one minute illumination with 20 mW/cm2 at 735 nm. A 730 nm long-pass filter was used to ensure that weak room light at shorter wavelengths was not reaching the sample. Red curve results from additional 15 minutes of illumination. Thus, most of the possible P700+ formation was achieved with fairly small illumination dose. Similar curves could be produced with illumination at 753 nm and 800 nm (Frame B). The Δ-Absorbance scale is the same in all frames of Figure 3 for easy comparison of data obtained for different samples and for different illumination wavelengths. Respective longpass filters were employed to ensure that P700+ formation was not due to the parasitic light of lower wavelengths. 3.2.2. Synechocystis sp. PCC 6803 Qualitatively similar results were observed also for Synechocystis. Illumination at 733 nm, 753 nm and 800 nm (again employing respective long-

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pass filters) resulted in the formation of persistent P700+ minus P700 hole spectra, as shown in Frame C in Figure 3. Required illumination doses were two orders of magnitude lower than those necessary to produce noticeable NPHB holes (see below), further supporting the idea that these light-induced features are associated with charge separation, and not NPHB. The main bleach is peaked at around 703 nm. However, this bleach is much broader than for T. elongatus, especially on the longer wavelength side. Other features of the P700+ minus P700 spectrum of Synechocystis sp. PCC 6803 also differ significantly from those reported above and for both T. elongatus and A. Platensis

21

. Differences in the P700+ minus P700 hole spectra between the

species indicate significant structural differences in the vicinity of the electron transfer chain (Figure 1A). Sharp higher-energy spectral features could be due to the electrochromic shifts of either the pigments of the RC or of the antenna pigments located close enough to the P700+. Figure 3D depicts the hole spectrum of a PR-type sample of the menB mutant (blue), that closely resembles the wild-type spectra. The main difference is that the features in the 674-686 nm region are much less intense in the case of the mutant.

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Figure 3. Frame A: T. elongatus PR sample. Green: difference between two consecutively measured absorption spectra. Blue: result of exposure of the sample to dim room light (several nW/cm2) for one minute. Black: after one minute of illumination at 733 nm with 20 mW/cm2. Red: after 15 minutes of 733 nm illumination. Dotted line indicates the cut-off wavelength of a long-pass filter, 730 nm protecting the sample from shorter-wavelength light. Frame B: T. elongatus; PR sample; NPHB-free P700+ minus P700 spectra produced with illumination at 800 nm. Frame C: Synechocystis sp. PCC 6803 PR sample NPHB-free P700+ minus P700 spectra produced with illumination at 733 nm (red), 753 nm (green) and 800 nm (black). Frame D: P700+ minus P700 spectrum of the Synechocystis sp. PCC 6803 menB mutant (blue).

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Figure 4. Frame A: Fluorescence excitation spectra of the partially reduced (PR) T. elongatus sample, cooled down to 5K in the dark. Emission (all wavelengths longer than 750 nm combined) was detected through a long-pass filter. Black to green curves resulted from consecutive scans from 735 nm to 670 nm. Magenta curve was recorded after an attempt to measure a SHB action spectrum covering the 735 nm to 700 nm range. Spectra are distorted (longer-wavelength range is amplified) due to the wavelength dependence of the transmission of the neutral density filters used for beam power attenuation. The spectra were deliberately left uncorrected to present their evolution in the longer-wavelength range more clearly. Frame B: An example of a two-component HGK produced at 706 nm with 300 µW/cm2.

3.3.3 Fluorescence Excitation Mode (T. elongatus) Manifestations of the ongoing P700 oxidation in partially reduced sample could also be seen in fluorescence excitation mode (Figure 4), with detection at wavelengths longer than 750 nm. Tuning the laser wavelength over the Qy absorption band (from about 735 nm to 670 nm) with light intensity of approximately 40 nW/cm2 results in a noticeable decrease of the signal for all excitation wavelengths, with the C719 band decreasing somewhat faster than others (note changing ratio of the 710 nm and 719 nm peaks; the spectra in Figure 4A were deliberately not corrected for transmission of the 16


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imperfect neutral density filters to enhance the long-wavelength region). Attempts to measure the hole growth kinetics (HGK) or the HB action spectra (hole depth dependence on wavelength for fixed illumination dose; usually employed to determine the shape of the site distribution function) had the same consequences. Qualitatively, these observations are consistent with the ongoing light-induced increase of the fraction of oxidized P700 that in turn reduces the fluorescence yield of the C719/F740 state. Its emission, peaked at 741 nm (Figure 2) is preferentially detected in fluorescence excitation mode experiments with a 750 nm long-pass filter. Observed HGK curves (example shown in Figure 4B) often contained at least two components, i.e., they were a superposition of (slower) NPHB and the (fast) initial decrease of the fluorescence signal likely due to the P700+ formation. 3.2.4. Amounts of Longer-Wavelength Light and Charge Separation upon Illumination beyond 800 nm. Since photo-oxidation of the P700 in a fraction of the complexes proved extremely easy in the partially reduced samples (i.e. it could be caused by illumination with nW/cm2 of broadband light for a minute), we had to estimate the amount of parasitic light (nonresonant fluorescence of the Ti-Sapphire crystal, scattered 532 nm pump laser light, etc) that might leak into the optical fiber between the laser system and the spectrometer and be responsible for unintended P700+ formation. The details of the estimation procedure are presented in the Supplemental Materials section. Summarizing, the amount of parasitic shorterwavelength excitation reaching the sample through the long-pass filters must not have exceeded 15 picowatts per cm2, all wavelengths combined. 15 pW/cm2 is two orders of magnitude lower than the intensity causing weak P700+ minus P700 signatures in Figure 3A (green) or three orders lower than in Figure 4A. It is also much lower than the intensity of the spectrophotometer’s beam. With typical illumination times of minutes, one cannot explain the

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P700 oxidation observed while the sample was illuminated at 733, 753 or 800 nm by parasitic light exposure. Thus, we are confident that P700 absorption extends to at least 800 nm for both species. This is in agreement with published results for T. elongatus and A. Platensis spinach and Nostoc punctiforme

20

21

and

. Thus, here we confirm the possibility of low-temperature

charge separation induced by long-wavelength excitation also for Synechocystis sp. PCC 6803. 3.2.5. Thermocycling of P700+ minus P700 features. Attributing various NPHB features in PSI requires detailed understanding of the behavior of the P700+ minus P700 signature, since the latter may contaminate the NPHB spectra. Figure 5A depicts the evolution of spectra resulting from thermocycling of the persistent P700+ minus P700 structure produced in T. elongatus PR (partially reduced) sample upon illumination at 733 nm. Black curve is the spectrum right after burning, at 5.5 K. This structure contains the signature of the P700 oxidation as well as a broad persistent hole at 733 nm. No resonant ZPH is observed for this illumination wavelength, at least at 0.2 nm resolution, as expected in the case of very strong electron-phonon coupling for the C719 state. By 60 K the persistent NPHB hole is recovered (see next sections), as in other pigment protein complexes, such as CP43

50

or Cytochrome b6f

51

for example. On the other

hand, significant fraction of the P700+ minus P700 signature persists (blue curve). Red and green curves were obtained after thermocycling to 115 K and 139 K, respectively. A small fraction of the 703 nm bleach persists up to these high temperatures. Careful examination of the data presented in Figure 5A and the spectra for other temperatures indicates that negative doublet at 684 nm and 687 nm recovers somewhat faster than other features associated with the 703 nm main bleach. The insert presents the recovery of the 703 nm bleach as well as its fit with Tmax

f = 1−

∫ g (T )dT , 0

18


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where g(T) is the distribution of barrier heights on a temperature scale, V(T)=kBTln(tmaxΩ0) is barrier height on the energy scale, tmax ≈ 60 sec is time spent at the peak cycle temperature T and

Ω0 is the attempt frequency on the order of 1012 Hz. Thus, ln(tmaxΩ0) ≈ 33. Two Gaussian components of g(T) are also presented. They are peaked at 65 K (1490 cm-1) and 140 K (3210 cm-1), respectively, and both have the width of about 55 K. This temperature dependence of the recovery of the P700+ minus P700 signature is in agreement with recombination of FA- and FB-, respectively, with about 14% fractions of both clusters being not pre-reduced in the beginning of the experiment. Figure 5B contains similar P700+ minus P700 thermocycling data obtained for Synechocystis sp. PCC 6803. The recovery of the 703 nm bleach exhibits two components with parameters very similar to those in T. elongatus, except the second, higher-temperature component attributed to FB- recombination is somewhat stronger. This corresponds to ~20% probability of each terminal iron-sulfur cluster not being successfully pre-reduced prior to burning. The tail of the 703 nm bleach in Synechocystis sp. PCC 6803 extends to 750 nm. The shape of this feature is preserved during thermocycling, meaning that it does not contain any NPHB component that would fully recover by the time thermocycling temperature reaches about 60 K. Same recovery behavior was observed for spectra produced with illumination wavelength of 753 nm. Most of the narrow shorter-wavelength features of the difference spectrum in both menB mutant and wild type samples recover faster than the 703 nm bleach, although they definitely persist past 60 K, the typical full recovery temperature for the non-photochemical spectral holes.

19



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Figure 5. Thermocycling of the P700+ minus P700 signature in partially reduced samples of T. elongatus (Frame A) and Synechocystis sp. PCC 6803 (Frame B). Hole spectra right after burning (black), after thermocycling to 55 K (blue), 105 K (red) and 139 K (T. elongatus) or 125 K (Synechocystis sp. PCC 6803) - green. Inserts depict the distributions of barriers deduced from the recovery of the main bleach at 703 nm.

20


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3.3. Persistent NPHB and the Signatures of Various Clusters of Strongly Coupled Pigments. To avoid contamination with the P700+ minus P700 signature, some of which always appears in the pre-reduced samples, measurements described in this subsection were performed using oxidized samples (O). Exposure to dim (~nW/cm2) room light, which was shown to cause detectable P700+ formation in the partially reduced samples, did not cause noticeable effects. Very weak P700+ minus P700 signature (similar in shape to the one shown in Fig. 3) could still occasionally be produced by an extensive illumination (~100 mW/cm2 for tens of minutes) at 733 nm through a 730 nm long-pass filter, indicating that not all P700 was perfectly oxidized. In these cases NPHB was explored after further illumination at 733 nm did not enhance this weak P700+ minus P700 signature. 3.3.1. T. Elongatus: Illumination at 725 nm is expected to probe only the C719/F740 state. The burning is very ineffective (black curve in Figure 6A), in line with the lifetime of C719/F740 state being shortened due to quenching by P700+

30

and the NPHB yield being reduced

accordingly, as well as with strong electron-phonon coupling resulting in low relative intensity of the ZPL. The NPHB spectrum presented here does not contain the 700+ minus P700 signature (see Figure 3A). The only clearly observable feature in the hole spectrum (black) in addition to the resonant hole and its NPHB anti-hole at about 710 nm is a positive one at 689.4 nm. This feature differs from the positive 691 nm feature of the P700+ minus P700 signature (grey). It most likely is the anti-hole of the higher excitonic component of the C719/F740 state. The higher excitonic state hole itself (at longer wavelength, probably around 695 nm) may be partially masked by the anti-hole of the original hole. The lower-energy hole / anti-hole pair appears to have larger intensity, in agreement with the C719/F740 state belonging to a chlorophyll cluster with a stronger lower exciton state, for instance A32-B7 (or A31-A32-B7-B6), see Figure 1B.

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The blue curve in Figure 6A is the hole spectrum obtained with λB=714.5 nm. Now both C715/F732 and C719/F740 states could be probed either directly or via phonon sidebands or via excitation energy transfer. However, it appears that the C719/F740 state contributes little to the blue curve. The copy of the λB=725 nm hole depicted in grey is deeper than the blue one beyond 720 nm. The C708 state is likely not probed at λB=714.5 nm. The electron-phonon coupling for the C708 state is weak, the absorption band is narrow, and therefore the origin of this state is not too far from 708 nm. Narrow zero-phonon lines observed in single-complex experiments

30,36-38

at around 710 nm likely belong to the C708 state. Zero-phonon hole at 714.5 nm is clearly visible, even at 0.25 nm resolution, indicating that the electron-phonon coupling for the C715/F732 state is weaker than for the C719 state. The 714.5 nm hole spectrum also exhibits features that do not resemble those of the P700+ minus P700 spectrum (Figure 3A). The broad positive peak at 703 nm most probably is the NPHB anti-hole of the longer-wavelength hole. Some of the features of the 725 nm and 714.5 nm hole spectra are similar (in particular – the positive feature at 689 nm), but two strong negative features at 670 nm and 682.5 nm are present only in the 714.5 nm hole spectrum. Thus, we propose that the higher excitonic bands of the C715/F732 state are located at 670 nm, 682.5 nm and possibly also around 695 nm. Figure 6B shows satellite hole structures obtained as a result of burning at 660 nm (c) and 532 nm (d). These satellite hole structures may contain contributions from all the red antenna states, if the energy is transferred to them effectively enough. The spectra are normalized to the same intensity of the lowest-energy hole, although they were almost normalized to begin with. The absorption around 532 nm is dominated by the carotenoids that then transfer energy to the chlorophylls. Both hole spectra clearly possess bleaches at 709 nm, matching the respective peak in the absorption spectrum (C708). This component of the satellite hole structure was present for

22


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lower illumination doses as well, indicating that C708 is always a weak energy trap at low temperatures, not only after the C719/F740 or C715/F732 states are burnt (i.e. blue-shifted by NPHB). This is in agreement with the SPCS results exhibiting narrow C708 emission lines around 710 nm 29,36-38.

Figure 6. T. elongatus with mostly oxidized P700 (sample O). Some spectra are shifted vertically for clarity. Frame A: Black, (a): hole spectrum after illumination at 725 nm, probing only the C719 state. Blue, (b): hole spectrum after illumination at 714.5 nm, probing C715 and C719 states. Grey: a shifted replica of the black curve. Frame B: Hole spectra for λB of 660 nm (magenta; c) and 532 nm (green, d). Orange, (c-d): the difference between curves (c) and (d), suspected hole spectrum of C712. Frame C: Hole spectrum for λB=532 nm : (green, d, same as in Frame B). Weighted sum of black and blue curves from Frame A : (red, a+1.5b). Frame D: Hole spectrum after burning at 660 nm (c, same as in Frame B, magenta) and the same spectrum 23



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after thermocycling to 40 K (e, brown). Light blue spectrum (c-e) is the difference spectrum representing thermocycling-induced recovery. A prominent difference between the hole spectra produced with excitation at 532 nm and 660 nm is that the 710 nm and 719 nm holes are poorly separated in the former hole spectrum. The difference spectrum (c minus d, orange curve) in Figure 6B exhibits the lowest-energy hole at 712 nm. Suppose that one could still assign this hole to either the C715 or C708 states. Comparison with the curve (b) in Figure 6A shows that the C715 signature and (c minus d) in Figure 6B are fairly dissimilar. The undisturbed isolated signature of the C708 state is somewhat difficult to obtain. Still, one could compare the hole spectrum for burn wavelength of 532 nm (green curve in Figure 6B, replicated in Figure 6C) and the weighted sum of the curves for λB=714.5 nm and 725 nm (red curve in Figure 6C). The former contains contributions from the C709, C715 and C719 states, and none or very little from the proposed new 712 nm state, while the latter contains contributions only from the C715/F732 and C719/F740 states. The C708 signature (that at least in the 650-690 nm region must resemble the difference between two curves in Frame C; longer wavelength region may be distorted by NPHB anti-holes of the lower states) would contain intense negative features at 670 nm and 682 nm, much more intense than those in the C715 signature (blue curve in Frame A). These 670 nm and 682 nm features appear stronger than the C708 hole itself, suggesting that the C708 state originates from one of the chlorophyll groups for which the weaker lower exciton state is predicted. These strong 670 nm and 682 nm features are definitely absent in the orange (c minus d) curve in Figure 6B. Thus, the orange curve in Figure 6B is different from either C708 or C715 signatures. Upon thermocycling the positive feature at 690 nm is recovering together with the 712 nm hole (see Figure 6D) and

24


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thus likely represents the higher-excitonic component of the respective chlorophyll cluster. Note that curve (c-e) in Figure 4D also includes some recovery of C715 and C719.

Figure 7: Hole spectra obtained at 5 K for Synechocystis sp. PCC 6803. Numbers indicate illumination wavelength. Black: 715 nm; green: 532 nm; magenta: 660 nm. Also shown for reference purposed are the P700+ minus P700 spectrum (grey curve) and absorption spectrum (dashed red curve). 3.3.2. Synechocystis sp. PCC 6803. For Synechocystis the situation is somewhat simpler due to the smaller number of the low-energy hole features produced by higher-energy illumination. Results are presented in Figure 7. The C714 hole, peaked at 713 nm here, is clearly visible in the hole spectrum for 655 nm excitation (magenta). The broad non-resonant hole due to C706 state has never been observed. Interestingly enough, the hole spectrum for excitation at 532 nm (green), via carotenoids, contains much weaker, if any, contribution from C714. Thus, in this respect C714 of Synechocystis sp. PCC 6803 appears similar to the newly-proposed C712 state of T. Elongatus. The satellite hole structures associated with these states are otherwise not that similar, though. The structure for C714 state (black) more closely resembles that of the C719

25



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state of T. elongatus. The green curve (λB=532 nm) still contains prominent higher-energy features. The shapes of both green and magenta curves strongly suggest the presence of some chlorophyll clusters with the lowest-energy state being very weak. The emission spectrum produced with 532 nm excitation does not contain enhanced higher-energy bands (in addition to the main peak at about 720 nm) that could correspond to the single-molecule ZPL distributions observed at 699 nm (allegedly analogous to C708 of T. Elongatus) and 710 nm 39. Thus, the lowest-exciton states of the respective Chl a cluster may be located in the red state region and their emission may be hidden under the C714 emission.

3.4. Thermocycling with Persistent NPHB Spectra Figure 8A summarizes the data on the recovery upon thermocycling of NPHB holes burnt into the lowest-energy states of T. Elongatus and Synechocystis sp. PCC 6803. These holes were presented in Figures 6 and 7. Relatively large error bars result from poor signal to noise ratio. The latter, in turn, is a result of poor hole burning yield due to the quenching of the red state excitations by P700+ or P700. The recovery of all lower-energy NPHB holes is governed by very similar barrier distributions and the holes fully recover by about 60 K. Thus, all pigment-protein complexes explored so far exhibit fairly similar barrier distributions, suggesting similar conformational changes are involved in NPHB and hole recovery. The evolution of the higherenergy features associated with the NPHB holes does not always follow the same simple pattern though. Some higher-energy features persist even after the lowest-energy one is recovered, occasionally some features experience shifts rather than recovery, etc. An example is given in Figure 8B. Note the blue shift of a positive feature around 686 nm (arrow). Complex evolution of the higher-energy features likely indicates the presence of the “domino effects” – NPHB of one pigment is associated with conformational changes propagating far enough to affect other 26


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pigments. This observation is in line with switching between different EET pathways leading to different emitting states observed in PSI using single complex spectroscopy at helium temperatures 43.

Figure 8: Frame A: recovery upon thermocycling for T. elongatus, λB =715 nm (black); T. elongatus, λB =725 nm (red); and Synechocystis sp. PCC 6803 λB =715 nm (green). Frame B: an example of the hole spectra during thermocycling-induced recovery. Blue: the 715 nm hole of Synechocystis sp. PCC 6803 at 5 K prior to thermocycling experiment. Red: hole spectrum after cycling to 47 K. Arrow indicates the 686 nm positive feature that is shifting without changing its magnitude. 4. Discussion. Before engaging in discussion on the NPHB signatures of the chlorophyll clusters responsible for the “red states”, we present some general considerations affecting this type of spectra. The following somewhat simplified discussion is based on several assumptions: First, we assume that the lowest-energy pigment in the coupled system is the one predominantly experiencing NPHB due to its longest lifetime. Second, we consider only the large NPHB-related spectral shifts corresponding to the higher hierarchal tiers of the protein energy landscape. Small (e.g., about 10

27



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cm-1 or 0.5 nm at around 700 nm) site-energy shifts unlikely change the status of the pigment as the lowest-energy one in the cluster, and subsequent acts of excitation likely result in the return of the burnt molecule to its original site energy (i.e. there would be no net persistent hole). In PSI this line of reasoning is important not only for the case of the higher-energy excitation followed by EET to the lowest state. Due to the strong electron-phonon coupling, the vast majority of the systems excited when burning directly into the lowest-energy states are still excited via their phonon sidebands, while their ZPL are located at longer wavelengths. One may compare spectral shifts in the subsequent discussion with the width of the PSI chlorophyll Qy band, around 30 nm. Figure 9 depicts the results of simplified modeling of NPHB spectra for several groups of chlorophylls usually considered the origin of the red antenna states. Ten thousand Chl a clusters were randomly generated, utilizing site energy distributions with the width of 180 cm-1 (8.6 nm) and peaked at the wavelengths reported by Byrdin et al.34. The energies and oscillator strengths of the excitonic states were determined by finding the eigenvalues and eigenvectors of the Hamiltonian matrix with site energies as diagonal elements and couplings as off-diagonal ones. The site energies were subject to disorder, off-diagonal energy disorder was not included into this simple model. Inclusion of small amount of off-diagonal disorder does not change which of the excitonic states of the cluster is stronger. Besides, limiting calculations to small clusters probably has larger effect than not including off-diagonal disorder. Inter-pigment couplings were from Ref 34 as well. Refined calculations

40

yield somewhat different couplings, but their signs

were obviously preserved as pigment orientations did not change in the refined structure 41, and the general shape of the spectra of the respective clusters must be preserved as well. NPHB was simulated by adding values randomly picked from a Gaussian distribution with peak of 100 cm-1 (~5 nm) and width of 18 cm-1 (~1 nm) to the site energies of the lowest-energy pigments in the

28


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cluster and recalculating eigenvalues and eigenvectors. Note that the higher excitonic states also shift, producing hole / anti-hole structures. Figure 9A depicts results for the B6-B7-A31-A32 cluster. As can be seen, the higher excitonic components of the NPHB spectrum are relatively weak. Thus, this shape is compatible with attributing the relatively featureless hole spectrum dominated by the C719 state in Figure 6A (black; λB=725 nm) to the B6-B7-A31-A32 cluster. Achieving better agreement will require some adjustment of the site energies with respect to those reported in Ref 34, and possibly also inclusion of additional relatively weakly coupled pigments into the cluster under consideration. Figure 9B depicts the NPHB spectrum expected for the B31-B32-B33 trimer. Red-shifting one of the pigments by 200 cm-1 (~9 nm) leads to some enhancement of the higher-energy states due to increased localization, although they remain fairly weak. Note that despite large differences in site energies of the participating pigments, significant redistribution of the oscillator strength is still taking place. Same is true also for A38-A39 or B37-B38 dimers whose higher-energy excitonic states are most intense, according to calculations. As there are multiple indications that A38-A39 does not contribute to the red state region

40,60

, only the results for the B37-B38 dimer are shown in Figure 9C and D.

The strongest redistribution of oscillator strength between the delocalized states occurs when the initial site energies of the chlorophylls in the cluster are close to each other. In Figure 9C the spectra were calculated for identical pre-burn site energies of the two chlorophylls (694 nm) 34. In Figure 9D the pre-burn site energy of B37 was shifted by 200 cm-1 (~9 nm) to the red. Note that the resulting change of the relative intensities of the higher- and lower-energy NPHB features is not very large. Summarizing, adjustments of the site energies do not qualitatively change the shapes of the NPHB spectra and one can be confident that simple simulations described here can be used to limit the circle of possible candidates for a given “red state” based

29



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on relative intensities of various features in the satellite hole structures. Explaining complex NPHB structures with several intense higher-energy features either requires assuming that more than one red state are excited simultaneously or one must include additional chlorophylls in the modeling of the respective clusters. In a tightly-packed pigment network like PSI, one should also consider a possibility that one small structural change triggered by optical excitation may affect transition frequencies of more than one pigment, and in this sense several pigments may experience NPHB caused by one photon. Neither of the long-λB NPHB spectra in Figure 6A suggests that the higher excitonic state of the respective chlorophyll cluster is much stronger than the lower-energy state. Thus, both A38A39 and B37-B38 dimers unlikely are the origin of either C719/F740 or C715/F732 states. B31B32-B33 may still be the origin of C715, as was first proposed in Ref 38, while B37-B38 could be the origin of C708, exhibiting strong higher excitonic bands. According to our results, energy difference between higher and lower-energy bands belonging to the same group of chlorophylls is significantly larger than twice the inter-pigment coupling (e.g. ~700 cm-1 versus twice the 200 cm-1 coupling for the C715/F732 state). This indicates that the site energies of respective chlorophylls (in the absence of inter-pigment interactions) are significantly different, and the C715/F732 state is somewhat localized on the lower-energy pigment of the respective chlorophyll Chl cluster. However, modeling shows that the difference in oscillator strengths of the lower and higher excitonic states is still comparable to that predicted in Ref 34.

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Figure 9: Calculated pre-burn (black), post-burn (blue) and difference/hole (red) spectra of chlorophyll Chl clusters likely giving rise to the red antenna states; unless otherwise specified site energies are according to Byrdin et al.34 unless specified otherwise. Frame A: B6-B7-A31A32 tetramer; Frame B: B31-B32-B33 trimer. Frame C: B37-B38 dimer with identical preburn site energies; Frame D: B37-B38 dimer with B37 site energy shifted to the red by 200 cm-1 (~9 nm); See Figures 1B or 10B for structures and locations of the above clusters within PSI. 31



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Page 32 of 45

The difference in the hole spectra for 532 nm and 660 nm excitation (see Figure 6B) can be considered as preliminary evidence for still another red antenna state in T. Elongatus. The combined intensity of the red region in T. Elongatus PSI is equal to that of 8-9 Chls per PSI monomer (i.e. per 96 chlorophyll molecules)

17

, and not all the oscillator strength of the

respective groups of strongly coupled chlorophylls is expected to be concentrated in the red state region; some fraction of the oscillator strength must still belong to the higher exciton states 32-35 (see Figure 9). Thus, the presence of a fourth red antenna state in T. elongatus PSI is in principle possible. Apparently, excitation via carotenoids at 532 nm does not bring the transferred energy into the vicinity of this cluster as effectively as the 660 nm excitation does. The distances between the chlorophylls and the carotenoids of PSI based on structure data

1,2

were reported in

Ref 34. In the PSI core carotenoids are distributed relatively uniformly and only a small fraction of the chlorophylls are further away from the nearest carotenoid than 7Å. Of the 90 antenna chlorophylls, 60 are in π−π stacking interaction with carotenoids 61. None of the clusters usually proposed as the origin of the red antenna states is composed of the chlorophylls that are consistently very far from any carotenoids. There are two clusters that exhibit sufficiently strong couplings and are located far from carotenoids, the A36-A37 and B14-B15 dimers

34

. These

chlorophylls, as well as the carotenoids, are highlighted in Figure 10A. Both A36-A37 and B14B15 dimers are located on the luminal side of the membrane and feature dipole-dipole couplings in the −120 cm-1 to −150 cm-1 range (or even stronger according to 40). For A36-A37 the lowerenergy component is expected to contain almost all the oscillator strength of the dimer, while for B14-B15 the lower state is expected to be the weaker one. With the shape of the satellite hole structure presented in Figure 6B (orange curve), and taking into the account that upon

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thermocycling the 712 hole is recovering together with the stronger positive feature at 690 nm, B14-B15 appears a more likely candidate for the new C712 state. In this respect it is worth noticing that B15 contribution to one of the lowest-energy states was suggested based on refined structure and improved calculations also in 40.

Figure 10. Frame A: Two dimers of sufficiently strongly coupled chlorophylls (red) that are located the farthest away from the carotenoids (yellow) and could be the origin of the C712 state. Frame B: original proposal of red antenna clusters in PSI 1,2, from Figure 1. In view of the above observation of some features in NPHB spectra persisting after the recovery of the lowest-energy NPHB holes and/or shifting (see Figure 8B) one could question the reliability of making conclusions about the relationships between particular pigments and red antenna states based on the NPHB data. The answer to this question is two-fold. First, thermocycling experiments as reported here are specifically useful in this respect as they allow for determining which parts of the spectra recover together and thus represent parts of the same structures (e.g. higher and lower excitonic states of the same chlorophyll cluster, see Figure 6D). Second, if some higher-energy features of the hole spectra represent the results of the “domino effects” (conformational changes propagating into the vicinity of pigments not participating in 33



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the cluster whose chlorophylls are excited) and not the higher-excitonic components of the “red states”, their relative intensities still could be used to make at least qualitative conclusions about the oscillator strengths of the lowest states. For instance, if one is to exclude the shifting feature at about 686 nm in Figure 8B from the “signature” of the C714 state in Synechocystis sp. PCC 6803, the latter will still indicate that the lower-energy excitonic component for the respective chlorophyll cluster is the strongest one, in agreement with assigning this state to the B7-A32 cluster.

5. Conclusions. We confirmed the results of Schlodder et al.21 and demonstrated that oxidation of P700 can occur upon illumination at up to 800 nm even at cryogenic temperatures also for Synechocystis sp. PCC 6803. Thus, this phenomenon appears to be universal for PSI, across species, and also a part of the wider pattern with CT states serving as primary electron donors also in PSII

18,19,22,23

. Achieving better control over oxidation state of the P700 in PSI samples

will be essential for improved NPHB measurements, including both resonant (such as HGK measurements for the purpose of protein energy landscapes exploration) and non-resonant burning. Specifically, using samples with perfectly reduced Fe4S4 clusters is strongly preferable as these samples will exhibit higher NPHB rate for the lowest state. We succeeded in obtaining the isolated NPHB signatures of the C719/F740 and C715/F732 states of T. elongatus as well as the C714 state of Synechocystis sp. PCC 6803, including their higher excitonic components. Additionally, we present preliminary evidence for the fourth red antenna state in T. elongatus, with the lower-energy absorption band peaked at 712.5 nm and likely belonging to chlorophylls not easily excited via energy transfer from the carotenoids. B15 and B14 chlorophylls likely

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contributed to this state. Obtaining separate satellite hole signatures of various red states allows for narrowing the range of possible site energies, and provides additional constraints for structure-based modeling of various optical spectra. Improved modeling of the NPHB data may require not only the adjustment of the site energies, but also inclusion of additional pigments into the coupled Chl a clusters being considered, as well as of the “domino” effects: spectral shifts of some pigments due to conformational changes triggered by the excitation of other pigments. The NPHB data, including that obtained in thermocycling experiments, represents one component of the extensive set, also including absorption, CD, MCD, circularly-polarized emission spectra

62

and time-domain and frequency-domain EET data, that should be subjected to global analysis to arrive to the final assignments of spectral bands in PSI (and other complexes) to particular chlorophylls. Interestingly, many of the above hole structures appear to possess similar features at similar wavelengths. Although it might be just a coincidence (it is difficult to fit the absorption bands of 96 interacting chlorophylls into a relatively narrow spectral region and not to observe some similar spectra), it is tempting to speculate that the red antenna states having higher exciton states in resonance with the absorption bands of the RC pigments might have some evolutionary advantage, by increasing the probability of EET from the respective chlorophylls to the P700. The EET models going beyond conventional Förster theory and describing EET between groups of strongly coupled pigment molecules

63-65

involve the overlaps between the whole densities of

states (including the higher exciton levels) rather than the overlaps between the emission and absorption spectra of the regular Förster theory.

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Acknowledgments: Concordia researchers thank NSERC, CFI and Concordia University. This work was also supported by the National Science Foundation Grant No. MCB0417142 to PF.

Supplementary Materials: Estimating the amount of parasitic light that could possibly reach the sample and cause unintended P700 oxidation.

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