far-red absorption and light-use efficiency trade-offs in ...10.1038... · far-red absorption and...

Articleshttps://doi.org/10.1038/s41477-020-0718-z

Far-red absorption and light-use efficiency trade-offs in chlorophyll f photosynthesisVincenzo Mascoli, Luca Bersanini and Roberta Croce ✉

Department of Physics and Astronomy and Institute for Lasers, Life and Biophotonics, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. ✉e-mail: [email protected]

SUPPLEMENTARY INFORMATION

In the format provided by the authors and unedited.

NatuRe PLaNts | www.nature.com/natureplants

http://orcid.org/0000-0003-3469-834X

mailto:[email protected]

http://www.nature.com/natureplants

1

650 700 750 800-200

-100

0

100

200

300

680 7200

2

4

DAS

λ (nm)

7 ps 41 ps > 5 ns

400 500 600 700 8000,0

0,5

1,0

1,5

Abso

rban

ce (a

.u.)

λ (nm)

WL-PSI FRL-PSI

680 720 760 800 8400,0

0,2

0,4

0,6

0,8

1,0

Nor

m. F

luor

esce

nce

λ (nm)

PSI WL PSI FR

5ps 17ps 51ps 135ps 3.3ns

700 750 800 850

-400

0

400

800700 750 8000

20

40

DAS

λ (nm)

Supplementary Figure 1. Spectroscopic data of isolated Photosystem I from CT.

A) 2D color map representing TRF data of PSI from WL-adapted CT excited at 400 nm and detected in the Chl Qy region with a Streak Camera setup (with a repetition rate of 250 kHz, an excitation power of 150 μW, the emission polarization set at magic angle with respect to the excitation, the temperature set to 10°C and a 400-ps time window).

FRL-PSI

WL-PSI A)

679.5 nm

B)

C) D)

E) F)

2

B) DAS from global analysis of TRF data in (A). The inset shows a magnification of the small long-lived component.

C) Absorption spectra (normalized at Qy maximum) of WL- and FRL-PSI of CT purified via sucrose gradient as described in the Materials and Methods section.

D) Time-integrated fluorescence spectra of PSI particles isolated from WL- (black lines) and FRL- (red lines) adapted cells of CT. The time-integrated spectra of WL PSI are obtained from the DAS in (B) (excluding the small long-lived component representing unconnected chlorophylls). The time-integrated spectra of FRL-PSI are calculated from the DAS in (F) (again excluding the minor long-lived component).

E) 2D color maps representing TRF data of PSI from FRL-adapted CT (same experimental conditions as in (A)).

F) DAS from global analysis of TRF data in (E). The first three components (black, red and blue DAS) describe energy transfer events following Chl excitation, whereas the fourth component (green DAS) describes trapping at FRL-PSI reaction centers. The small long-lived DAS (which is magnified in the inset) is due to small fractions of energetically-uncoupled pigments (and, possibly, PSII). See Supplementary Figure 2 for an overlay of the raw data with the fitted traces. The same experimental conditions were used for the correspondent measurements on PSI of CF shown in Figure 1 of the manuscript.

For each sample, the data are representative of two different preparations yielding similar results.

3

0 100 200 3000

400

800

1200

Fluo

resc

ence

cou

nts

Time (ps)

690 fit 690 720 fit 720

CF WL-PSI

0 100 200 3000

50

100

150

200

250

300

Fluo

resc

ence

cou

nts

Time (ps)

690 fit 690 720 fit 720

CT WL-PSI

0 100 200 3000

200

400

600

800

1000

Fluo

resc

ence

cou

nts

Time (ps)

685 685 fit 745 745 fit 790 790 fit

CF FRL-PSI

0 100 200 3000

200

400

600

800

1000

Fluo

resc

ence

cou

nts

Time (ps)

685 685 fit 750 750 fit 790 790 fit

CT FRL-PSI

Supplementary Figure 2. Time-resolved fluorescence (TRF) measurements of PSI isolated from WL- and FRL- adapted strains. A,B) Overlay of raw and globally fitted fluorescence time traces at selected emission wavelengths from TRF data of WL-PSI of CF (see also Figure 1A-B) and WL-PSI of CT (see also Supplementary Figure 1A-B). The selected emission wavelengths highlight the contributions from the two main spectral forms, i.e. bulk Chls a (690 nm) and the red Chls a (720 nm). C,D) Overlay of raw and fitted fluorescence time traces at selected emission wavelengths from TRF data of FRL-PSI of CF (see also Figure 1E-F) and FRL-PSI of CT (see also Supplementary Figure 1E-F). The traces show how the initially excited Chl a (represented by the black fluorescence trace at 685 nm) quickly decays due to energy transfer to Chl f, whose rise can be observed at 745-750 nm (black and red DAS’s in Figure 1 of the manuscript). The signal at 790 nm due to red-shifted Chl f builds up on a longer timescale due to slower equilibration with the other Chls f at 745-750 nm (blue DAS’s in Figure 1F and Supplementary Figure 1F). For each sample, the data are representative of two different preparations yielding similar results.

A) B)

C) D)

4

400 500 600 700 8000,0

0,5

1,0

1,5Ab

sorb

ance

(a.u

.)

λ (nm)

FRL-PSII

640 680 720 760 800 8400,0

0,2

0,4

0,6

0,8

1,0

Nor

m. F

luor

esce

nce

λ (nm)

400 nm 440 nm 500 nm 580 nm

0.06ns 0.41ns 1.47ns 3.11ns

680 700 720 740 7600

1

2

3

λ (nm)

DAS

(a.u

.)

Open PSII 0.04 ns 0.16 ns 0.57 ns 1.54 ns 4.00 ns

680 700 720 740 760

0.0

0.5

1.0

1.5

DAS

(a.u

.)

λ (nm)

Closed PSII

0 2 4 6 80,0

0,2

0,4

0,6

0,8

1,0

Nor

m. F

luor

esce

nce

Time (ns)

IRF Open Closed

740 nm emission

680 700 720 740 7600,0

0,5

1,0

1,5

2,0

2,5

λ (nm)

open closed

Aver

age

Life

time

(ns)

Supplementary Figure 3. Fluorescence data of isolated FRL-Photosystem II from CT.

A) Absorption spectrum of PSII particles isolated from FRL-adapted CT.

B) Normalized fluorescence spectra of PSII particles from FRL-adapted CT excited at different wavelengths. Beside the major emission band peaking at 746 nm and stemming from FRL-PSII, a second band (peaking at 683 nm) in the Chl a region can be observed, which results from the presence of WL-PSII

674 nm

A) B)

C) D)

E) F)

5

in the preparation. This suggests that this strain retains some WL-PSII after adaptation to FRL, as recently observed also for another FaRLiP strain1. Upon 580 nm excitation, the emission band in the far-red blue-shifts due to an increased contribution in the 720-nm region. This observation can be explained by the presence of FRL-PBS, whose bicylindrical core might stick to PSII in the preparation1,2.

C,D) DAS from global analysis of TRF data on PSII particles isolated from FRL-adapted CT with nearly open (C) and closed (D) reaction centers. The data were recorded with a TCSPC setup exciting at 438 nm. FRL-PSII with open RCs was measured at 0.2 μW (2.5 MHz repetition rate) and upon addition of ferricyanide 0.4 mM, (for each wavelength, the fluorescence trace was recorded for 10 minutes) whilst FRL-PSII with closed RCs was measured at 50 μW (10 MHz repetition rate) after addition of 50 μM DCMU (each trace recorded until reaching 10000 counts at peak maximum). An oxygen scavenging mixture consisting of catalase (50 mg/ml), glucose oxidase (100 mg/ml) and glucose (5 mM) was used to increase sample stability. The temperature was set to 8 °C. After Chl excitation at low powers (where most RCs are open), the excited state kinetics from PSII particles of CT can be described by four components (C). The fastest (< 100 ps) DAS shows significant amplitude in the Chl a region, with a peak at 680 nm, compatible with the assignment to WL-PSII3,4. The 400-ps component, instead, most likely incorporates contributions from WL-PSII (in the visible region), FRL-PBS (around 720 nm) and FRL-PSII. The 1.5-ns DAS is entirely located in the FR region. The small long-lived component in (C) is due to a minor fraction of particles with closed RCs (see Supplementary Figure 4 for details). Notably, the 400-ps and 1.5-ns DAS have different shapes in the FR, that of the 400-ps component displaying a larger contribution at 720 nm, which is likely explained by the presence of FRL-PBS. On the other hand, in CF, where FRL-PBS are virtually absent in the FRL-PSII preparation (see emission spectra in Figure 2A), the 200-ps and the 1.0 ns DAS (Figure 2C) have very similar shape. Upon closure of the RCs (D), the excited state kinetics becomes extremely complex due to the sample heterogeneity. The average lifetime, however, increases both at 680 nm (where the amplitude of the longer-lived component increases with respect to the case of open RCs (C)), and in the FR (see also (E)). FRL-PSII of CT with closed RCs displays, as already observed for the case of CF, a strong 4-ns component. An overlay of the correspondent experimental and fitted TRF traces can be found in Supplementary Figure 5.

E) Normalized TRF traces of FRL-PSII particles from CT with open (black) and closed (red) RCs. Measurements were performed with a TCSPC setup upon 438 nm excitation. The instrumental response function (IRF) detected at 740 nm is shown in grey.

F) Average fluorescence lifetime of PSII from FRL-adapted CT as a function of wavelength (based on the DAS in (C,D). Due to the sample heterogeneity (WL-PSII, FRL-PBS and FRL-PSII coexist in the preparation), the average lifetime increases at longer wavelengths and peaks at 750 nm. This trend is maintained when PSII particles are measured in closed state.

Data are representative of two different preparations yielding similar results, the only difference being in the relative amount of FRL-PBS

6

0 2 4 60,0

0,2

0,4

0,6

0,8

1,0

Nor

mal

ized

fluo

resc

ence

Time (ns)

0.1 uW 0.2 uW 0.5 uW 1 uW 5 uW 50 uW IRF

CF

0 2 4 60,0

0,2

0,4

0,6

0,8

1,0

Nor

mal

ized

fluo

resc

ence

Time (ns)

0.1 uW 0.2 uW 0.5 uW 1 uW 5 uW 50 uW IRF

CT

0.1 uW

0.2 uW

0.5 uW 1 u

W5 u

W50

uW0,0

0,1

0,2

0,3

0,4

0,5

0,6

Ampl

itude

Fra

ctio

n

0.24 ns 1.10 ns 3.33 ns

0.1 uW

0.2 uW

0.5 uW 1 u

W5 u

W50

uW0.0

0.1

0.2

0.3

0.4

0.5

0.6 0.20 ns 1.33 ns 3.69 ns

720 740 7603.2

3.4

3.6

3.8

4.0

F m/ F

0

λ (nm)680 700 720 740 760

1.8

2.0

2.2

2.4

F m/ F

0

λ (nm)

Supplementary Figure 4. TRF of PSII particles isolated from FRL-adapted strains. A,B) TRF traces recorded at 740 nm (upon 438 nm excitation) at different excitation powers for both strains. The data were recorded with a TCSPC setup at 2.5 MHz repetition rate, unless at 50 μW power, where the repetition rate was 10 MHz. The temperature was set to 8 °C and ferricyanide 0.4 mM was used to keep RCs as much open as possible. The traces are nearly power independent below 0.5 μW and become increasingly longer-lived at higher powers. The instrumental response function (IRF) detected at 680 nm is shown in grey. C,D)

A) B)

C) D)

E) F)

7

Results from a simultaneous multi-exponential fit of the above traces. Three lifetime components were needed to satisfactorily fit the data and the depicted bars represent the fractional amplitude of each component at different excitation powers. In both strains, the increasing excitation pressure involves an increase of the long-lived (> 3 ns) component at the expense of the shorter ones. The >3 ns component is therefore assigned to PSII particles with closed RCs and its amount is vanishingly small below 0.5 μW, confirming that PSII with open RCs can be only measured at very low powers.

E,F) Ratio between the fluorescence lifetime measured in closed (Fm) and open state (Fo) conditions at different wavelengths for CF (E) and CT (F). Fm/Fo is calculated as the ratio between the average lifetimes measured in closed state and open state, plotted in Figure 2F (for CF) and Supplementary Figure 3F (for CT).

The power dependency in (A,B,C,D) was investigated once for each strain.

8

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)

720 720 fit Residuals

χ2 = 1.04

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


CF FRL-PSII open

χ2 = 1.04

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.04

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.02

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.02

CF FRL-PSII closed

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.02

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 0.98

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 0.98

CT FRL-PSII open

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 0.98

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.03

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.03

CT FRL-PSII closed

0 2 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.03

Supplementary Figure 5. TRF of PSII particles isolated from FRL-adapted strains. Overlay of raw and globally fitted TRF traces of PSII particles from FRL-acclimated CF and CT (measured with a TCSPC setup upon 438 nm excitation). For each line (representing a different dataset), three experimental time-traces are shown in grey corresponding to three selected emission wavelengths (specified in the legends). For each dataset, all traces detected at different wavelengths are globally analyzed as explained in the methods section obtaining the fitted traces (shown in black) and the global χ2 shown at each line. The corresponding residuals (i.e. the difference between experimental and fitted traces) are shown in blue. The DAS obtained from globally analyzing these data can be found in Figures 2C-D (for PSII of FRL-CF) and

9

Supplementary Figures 3C-D (for PSII of FRL-CT). For FRL-PSII of CF, the traces are representative of 2 technical replicas. For FRL-PSII of CT, the traces are representative of 2 different preparations.

680 720 760 800-1000

0

1000

2000

DAS

(a.u

.)

λ (nm)

14 ps 310 ps 3.5 ns

Supplementary Figure 6. TRF measurements of PSII particles isolated from FRL-adapted strains. DAS from TRF data of PSII particles from FRL-adapted CF measured with a Streak Camera setup with closed RCs (400 nm excitation, 50 μW power and 250 kHz repetition rate + 50 μM DCMU). The experimental time window for this experiment was 1.5 ns. The temperature was set to 8 °C. The black DAS represents Chl a to Chl f downhill energy transfer, whereas the red and blue DAS represent Chl excited state decay in presence of closed RCs. These data demonstrate that the amount of excitations retained by Chls a after few tens of ps is negligible. The data were measured once on each strain (data for PSII of CT not shown), with similar results and fully consistent with correspondent measurements with TCSPC (Figures 2 and S3).

680 720 760 800 8400.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

fluo

resc

ence

λ (nm)

CF FRL-PSI CT FRL-PSI CF FRL-PSII CT FRL-PSII

Supplementary Figure 7. Fluorescence spectra of FRL Photosystems. Normalized steady-state fluorescence spectra of FRL-PSI and PSII from CF and CT. The emission spectrum of FRL-PSI of CT (peaking at 752 nm) is 6.5 nm redshifted in comparison to that of FRL-PSI of CF (peaking at 745.5 nm). The emission spectrum of FRL-PSII of CT (peaking at 746.5 nm) is 8 nm redshifted in comparison to that of CF (peaking at 738.5 nm). The spectra are representative of 2 different preparations with similar results on each strain/sample.

10

400 500 600 700 8000.0

0.5

1.0

1.5

Abso

rban

ce (a

.u.)

λ (nm)

WL FRL

640 680 720 760 800 8400.0

0.2

0.4

0.6

0.8

1.0

Nor

m. F

luor

esce

nce

λ (nm)

400 nm 440 nm 580 nm

CT WL

640 680 720 760 800 8400.0

0.2

0.4

0.6

0.8

1.0

CT FRLN

orm

. Flu

ores

cenc

e

λ (nm)

400 nm 440 nm 580 nm

Supplementary Figure 8. Absorption and emission spectra of intact cells of CT. A) Absorption spectra of intact cells of CT adapted to WL (black lines) and to FRL (red lines). The Chl f contribution to the absorption spectrum of FRL-adapted cells can be observed at wavelengths > 700 nm. B) Emission spectra of intact cells of CT adapted to WL excited at different wavelengths. 440-nm excitation is selective for Chls, whereas 580-nm excitation is selective for the PBS. 400-nm light mostly excites Chls, with some smaller PBS contribution. C) Emission spectra of intact cells of CT adapted to FRL excited at different wavelengths. Data from WL-cells are representative of 2 biological replicas, those from FRL-cells of 3 biological replicas, all yielding similar results.

B) C)

A)

11

600 650 700 750-100

0

100

200

650 700 750 -0,2

-0,1

0,0

DAS

(a.u

.)

λ (nm)

8 ps 33 ps 160 ps 380 ps inf

CT WL - low power

600 650 700 750

-1000

0

1000

2000

DAS

(a.u

.)

λ (nm)

10 ps 31 ps 150 ps 840 ps

CT WL - closed PSII

9 ps 50 ps 170 ps 1.1 ns inf

650 700 750 800

-200

0

200

400

700 800 0

2

DAS

(a.u

.)

λ (nm)

CT FRL - low power

650 700 750 800-4000

-2000

0

2000

4000

6000CT FRL - closed PSII

DAS

(a.u

.)

λ (nm)

11ps 60ps 150 ps 410 ps 2.1 ns

Supplementary Figure 9. TRF measurements of intact cells of WL- and FRL-adapted CT.

The vanishingly small and flat orange component observed at low powers (and magnified in the insets), whose lifetime is much longer than the experimental time window, is needed for baseline correction. More details on the experimental conditions, as well as color maps representing the raw data, can be found in Supplementary Figures 10 and 12 and their captions. See Supplementary Figures 11 and 13 for an overlay of experimental/fitted TRF traces.

A,B) DAS from global analysis of TRF data of WL cells of CT measured with a Streak Camera setup upon 400 nm excitation at low power, were most PSII RCs are open (A), and with closed PSII (B). Under both conditions (open and closed PSII), the two fastest components (black and red DAS) are mostly related to PSI energy equilibration and trapping (which are insensitive to excitation pressure). In both (A) and (B), the blue DAS mostly stems from the PBS, with some smaller contributions from the photosystems, whereas the longer-lived green component, whose lifetime increases from 380 ps at low powers to 840 ps in closed state, can be ascribed to PSII.

C,D) DAS from global analysis of TRF data of FRL cells of CT at low powers (C) and with closed PSII (D). In both conditions (open and closed PSII), the black and red DAS represent subsequent energy equilibration steps: a faster transfer from Chl a to Chl f (black DAS), and a slower transfer from the Chl f pool (and possibly redshifted Chl a) to a Chl f species redshifted to 790-800 nm (red DAS), which for its timescale and spectrum can be assigned to FRL-PSI only (Supplementary Figure 1F). The blue component, whose lifetime (about 150 ps) and spectrum are not much sensitive to the state of PSII RCs, can be ascribed

A) B)

C) D)

12

mostly to FRL-PSI (see blue DAS in Supplementary Figure 1F). This component also includes some contribution from WL-PSII and WL-PBS (the latter are excited to a lesser extent at 400 nm) at shorter wavelengths. At the same time, a small dip at 720 nm is also observed in the 150-ps DAS (more visible when PSII RCs are closed, (D)), which suggests that the redshifted pigments in the FRL-PBS accept excitations on this timescale. The longest-lived component (1.1 ns) observed when PSII RCs are mostly open (green DAS in (C)) can be assigned to a mix of FRL-PSII and FRL-PBS (with some closed WL-PSII at shorter wavelengths). In closed state, the average lifetime in the FRL-PSII spectral region increases and two long-lived PSII related components can be separated (green and orange DAS in (D)). The shortest one (green DAS) is blue shifted and, therefore, is largely contributed by FRL-PBS, whereas the longest one (orange DAS) has an enhanced contribution from FRL-PSII. The longest lifetime that can be assigned to PSII in vivo is therefore in the order of 2 ns, which is shorter than that observed for PSII in vitro. The contribution from (closed) WL-PSII is also evident in the green and orange DAS. Data from WL-cells are representative of 2 biological replicas, those from FRL-cells of 3 biological replicas, all yielding similar results.

13

Supplementary Figure 10. TRF measurements of intact cells of WL-adapted strains. 2D color maps representing TRF data of WL-adapted cells of CF (A,B) and CT (C,D) excited at 400 nm and detected in the Chl Qy region (with a repetition rate of 250 kHz and an experimental time window of 1.5 ns). Each strain was measured under two different conditions, i.e. with an excitation power of 5 μW (low excitation pressure, meaning that most PSII RCs are open, left side) or with an excitation power of 20 μW after 50 μM DCMU addition (closed PSII, right side). Measurements were performed at room temperature (RT). For both strains, the emission at 680 nm becomes clearly longer lived at high excitation pressure due to RC closure. The DAS shown in Figures 4A-B and Supplementary Figures 9A-B are obtained by global analysis of the data presented here (corresponding to panels A,B,C,D, respectively). Data are representative of 2 biological replicas yielding similar results.

A) B)

C) D)

CF WL - low power CF WL- closed PSII

CT WL - low power CT WL- closed PSII

14

0 400 800 12000

50

100

150

200

250Fl

uore

scen

ce c

ount

s

Time (ps)

660 660 fit 681 681 fit 720 720 fit

CF WL - low power

0 400 800 12000

400

800

1200

1600

Fluo

resc

ence

cou

nts

Time (ps)

660 660 fit 681 681 fit 720 720 fit

CF WL - closed PSII

0 400 800 12000

50

100

150

200

250

Fluo

resc

ence

cou

nts

Time (ps)

660 660 fit 681 681 fit 720 720 fit

CT WL - low power

0 400 800 12000

400

800

1200

1600

Fluo

resc

ence

cou

nts

Time (ps)

660 660 fit 681 681 fit 720 720 fit

CT WL - closed PSII

Supplementary Figure 11. TRF measurements of intact cells of WL-adapted strains. Overlay of raw and globally fitted fluorescence time traces at selected emission wavelengths (see Supplementary Figure 10 for 2D color maps representing the entire raw datasets and Figures 4A-B and Supplementary Figures 9A-B for the DAS resulting from global analysis). The selected emission wavelengths highlight contributions from the phycobilisomes (660 nm), PSII (681 nm) and PSI (720 nm). Data are representative of 2 biological replicas yielding similar results.

A) B)

C) D)

15

A) B)

C) D)

CF FRL - low power CF FRL- closed PSII

CT FRL - low power CT FRL- closed PSII

CF FRL - low power (shorter time window)

CT FRL - low power (shorter time window) E) F)

16

Supplementary Figure 12. TRF measurements of intact cells of FRL-adapted strains.

A,B) 2D color maps representing TRF data of intact cells from FRL-adapted CF. Measurements were performed with a Streak Camera setup upon 400 nm excitation, 250 kHz repetition rate at low powers (5 μW, when most PSII RCs are open, (A)) and with closed PSII (50 μW + 50 μM DCMU, (B)). The time window is 1.5-ns long.

C,D) Same as for (A,B) for FRL-adapted cells of CT.

E,F) 2D color maps representing TRF data (measured with an experimental time window of 400 ps) of intact cells from FRL-adapted cells of CF (E) and CT (F). Measurements were performed with a Streak Camera setup upon 400 nm excitation, 250 kHz repetition rate, at low powers (5 μW, when most PSII RCs are open). All measurements are at RT.

The DAS shown in Figure 4C and Supplementary Figure 9C (i.e. at low powers) are obtained from global analysis of the 400-ps window datasets (E,F) in order to improve the overall time-resolution (see Materials and Methods). Analysis of the 1.5-ns window datasets yielded similar results (not shown). The DAS with closed PSII RCs in Figure 4D and Supplementary Figure 9D are obtained, instead, from global analysis of the 1.5-ns window datasets (B,D) for a better estimation/separation of the long-lived components. Comparison between the CF and CT related color maps highlights the higher relative amount of FRL-PSII in CF (the intensity of the long-lived PSII-related component is larger than in CT). The presence of some residual WL-PSII is more evident in CT than in CF (compare the 680-nm regions of the two color maps in the bottom panel). Data are representative of 3 biological replicas yielding similar results.

17

0 100 200 3000

200

400

600

800Fl

uore

scen

ce c

ount

s

Time (ps)

680 680 fit 740 740 fit 790 790 fit

CF FRL - low power shorter time window

0 400 800 12000

4000

8000

12000

16000

Fluo

resc

ence

cou

nts

Time (ps)

680 680 fit 740 740 fit 790 790 fit

CF FRL - closed PSII longer time window

0 100 200 3000

100

200

300

400

500

Fluo

resc

ence

cou

nts

Time (ps)

680 680 fit 750 750 fit 790 790 fit

CT FRL - low powershorter time window

0 400 800 12000

2000

4000

6000

8000

10000

Fluo

resc

ence

cou

nts

Time (ps)

680 680 fit 750 750 fit 790 790 fit

CT FRL- closed PSII longer time window

Supplementary Figure 13. TRF measurements of intact cells of FRL-adapted strains. Overlay of raw and globally fitted fluorescence time traces at selected emission wavelengths (see Supplementary Figure 12 for 2D color maps representing the entire raw datasets and Figures 4C-D and Supplementary Figures 9C-D for the DAS resulting from global analysis). The low power traces (A,C) are extracted from the 400-ps window datasets (Supplementary Figures 12E,F), whereas the traces with closed PSII (B,D) are extracted from the 1.5-ns datasets (Supplementary Figures 12B,D). The selected emission wavelengths highlight contributions from Chl a and WL-PSII (680 nm), FRL PSI/PSII (740-750 nm) and FRL PSI (790 nm). As already discussed for the fluorescence time traces from isolated FRL-PSI (Supplementary Figure 2), these traces show how the initially excited Chl a (represented by the black fluorescence trace at 680 nm) undergoes a quick decay due to energy transfer to Chl f in both PSI and PSII, whose rise can be observed at 740-750 nm (black DAS’s in Figures 4C-D and Supplementary Figures 9C-D). The signal at 790 nm due to red-shifted Chl f in PSI builds up on a longer timescale due to slower energy transfer from the other Chls f at 740-750 nm (red DAS’s in Figures 4C-D and Supplementary Figures 9C-D). Comparison between the CF and CT related traces also highlights the higher relative amount of PSII in CF (the residual long-lived PSII related component in the green trace is larger than in CT). In both strains, the 680-nm fluorescence does not decay entirely after few tens of ps as it was the case for isolated FRL-PSI (black trace in Supplementary Figures 2C-D). This can be explained by the presence of some residual WL-PSII (and 680-nm emitting PBS units). The presence of some residual WL-PSII is more evident in CT, where the relative intensity of the 680-nm (black) trace is higher. Data are representative of 3 biological replicas yielding similar results.

A) B)

C) D)

18

650 700 750 8000

4000

8000

12000

620 640

0

500

1000

Fluo

resc

ence

cou

nts

λ (nm)

CF FRL- closed PSII

Supplementary Figure 14. TRF measurements of intact cells of FRL-adapted CF. Reconstructed time zero spectrum (i.e. immediately after laser excitation) for the TRF data of FRL-acclimated cells of CF in closed state (measured with the Streak Camera setup). The spectrum, corresponding to the sum of all DAS shown in Figure 4D in the main text, is entirely positive and goes to zero in the region below 650 nm. Data are representative of 3 biological replicas yielding similar results.

720 760 800 8400.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

fluo

resc

ence

λ (nm)

Low P - 1.1 ns High P - 0.6 ns High P - 2.1 ns Isolated PSII

Supplementary Figure 15. Fluorescence spectral components of FRL-PSII. Normalized DAS/emission spectra of PSII-related components from CF. The black, red and blue normalized DAS are taken from Figure 4C-D: the black one from the measurement at low power (green DAS in Figure 4C), the red and blue from that in closed state (green and orange DAS in Figure 4D). For comparison, the steady-state fluorescence spectrum of FRL-PSII particles isolated from CF (Figure 2B) is displayed in dashed magenta. In vivo data are representative of 3 biological replicas yielding similar results. In vitro data are representative of 2 similar preparations.

19

0 1 2 30,0

0,2

0,4

0,6

0,8

1,0 0.2 µW 0.3 µW 0.5 µW 1 µW 2 µW 10 µW 50 µW 50 µW/DCMU IRFFl

uore

scen

ce

Time (ns)

CF WL

0 1 2 30.0

0.2

0.4

0.6

0.8

1.0

Fluo

resc

ence

Time (ns)

0.2 µW 0.3 µW 0.5 µW 1 µW 2 µW 10 µW 50 µW 50 µW/DCMU IRF

CT WL

Supplementary Figure 16. TRF measurements on intact cells of WL-adapted strains. Normalized fluorescence time traces detected at 680 nm upon 438 nm excitation of WL-adapted intact cells of CF (A) and CT (B) at increasing powers (at RT). The data were recorded with a TCSPC setup at 10 MHz repetition rate. All traces were recorded for 10 minutes or until reaching 2000 counts at peak maximum. The traces become longer-lived at increasing powers due to an increasing amount of closed PSII RCs. The traces are power-independent only at excitation powers below 0.5 μW, implying that the cells must be measured at very low powers to be kept in fully open state. The IRF detected at 680 nm is shown in grey.

An exponential fit of the fluorescence trace of WL-CT cells with open PSII RCs (0.2 μW) requires two lifetime components: 42 ps (73% amplitude) and 220 ps (27% amplitude), implying that the longest PSII related lifetime in fully open state is 220 ps, which is shorter than that measured with the Streak Camera setup at 5 μW (Supplementary Figure 9A). For the trace measured in closed state (50 μW + 50 μM DCMU), three lifetimes were needed: 41 ps (59% amplitude), 280 ps (15%) and 880 ps (26%). In both conditions, the shortest lifetime component is in the order of 40 ps and can be ascribed mostly to PSI trapping, with some contribution also stemming from PSII, whose fluorescence kinetics is known to be multi-exponential (indeed, the relative amount of the 40 ps-component decreases when closing PSII RCs). The slowest PSII-related component in closed state is therefore 880 ps in CT cells. The average lifetime at 680 nm for CT cells rises from 90 ps in open state to 300 ps in closed state, implying Fm/Fo = 3.3.

Fitting of the fluorescence trace of WL-CF cells with open RCs (0.2 μW) needed three lifetime components: 45 ps (75%), 220 ps (23%) and 1.2 ns (2%). The vanishingly small 1.2 ns component can be ascribed to a minor fraction of PSII with closed RCs. Again, the longest lifetime that can be attributed to PSII with open RCs is 220 ps (which is shorter than that that reported in Figure 4A for the Streak Camera measurement at low power). When closing PSII RCs with DCMU and higher excitation power, three lifetime components are also needed: 43 ps (59%), 430 ps (19%) and 1.3 ns (22%). The shortest lifetime component (still around 40 ps) can be interpreted in a similar fashion as for CT cells (i.e. it is mostly due to PSI trapping, with some contribution from PSII), whereas the slowest PSII-related component in closed state rises to 1.3 ns in CF cells. The average lifetime at 680 nm for CF cells rises from 86 ps in open state (excluding the 2% 1.2-ns component) to 390 ps in closed state, resulting in Fm/Fo = 4.6.

An overlay of the experimental and fitted TRF traces can be found in Figure S16. The power-dependency was investigated once for each strain, while the traces used for global analysis are representative of 3 technical replicas (see Figure S16) and are consistent with the corresponding TRF data measured with the Streak Camera (Figure 4A-B and Supplementary Figure S9A-B).

A) B)

20

0,0 0,5 1,0 1,5

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 1.00

CF WL-cells open

Fluo

resc

ence

(a.u

.)

Time (ns)


0 1 2 3

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 0.99

CF WL-cells closed

Fluo

resc

ence

(a.u

.)

Time (ns)


0,0 0,5 1,0 1,5

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 0.62

CT WL-cells open

Fluo

resc

ence

(a.u

.)

Time (ns)


0 1 2 3

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 0.93

CT WL-cells closed

Fluo

resc

ence

(a.u

.)

Time (ns)


Supplementary Figure 17. TRF measurements on intact cells of WL-adapted strains. Overlay of raw (grey lines) and fitted (black lines) TRF traces of WL-acclimated CF and CT (measured with a TCSPC setup upon 438 nm excitation). The emission wavelength is specified in the legends. For each trace, the χ2 and residuals (blue lines) resulting from the exponential fitting are also shown. The results from global analysis of these data can be found in the caption of Supplementary Figure 16. For each sample, the traces are representative of 3 technical replicas with similar results.

21

0,0 0,5 1,0 1,5 2,0 2,5 3,00,0

0,2

0,4

0,6

0,8

1,0

Nor

mal

ized

fluo

resc

ence

Time (ns)

0.2 uW 0.5 uW 2 uW 50 uW 50 uW + DCMU IRF

CF FRL

740 760 780

0,0

0,5

1,0

DAS

λ (nm)

0.04 ns 0.13 ns 0.41 ns 1.0 ns

CF FRL - open PSII

740 760 780

0,0

0,2

0,4

0,6

DAS

λ (nm)

0.047 ns 0.18 ns 0.99 ns 2.0 ns

CF FRL - closed PSII

Supplementary Figure 18. TRF measurements of intact cells of FRL-adapted CF. A) Normalized TRF traces detected at 735 nm upon 438 nm excitation of cells of FRL-adapted CF at increasing powers (at RT). The IRF trace is shown in grey. The emission wavelength was chosen to enhance PSII contribution. Measurements were performed with a TCSPC setup at 10 MHz repetition rate. All traces were recorded for 10 minutes or until reaching 4000 counts at peak maximum. The traces become longer-lived at increasing powers due to an increasing amount of closed PSII RCs. The traces are power-independent at excitation powers below 2 μW. Due to the spectral heterogeneity of FRL-adapted cells, fluorescence traces detected at three distinct wavelengths (735, 750 and 790 nm) were used to perform the following global exponential fit (B,C).

B,C) Global analysis of TRF data of FRL-CF cells in open state (2 μW, (B)) and in closed state (50 μW + 50 μM DCMU, (C)) upon 438 nm excitation. Four components were required for a satisfactory global fit under both conditions. In open state, the relative amplitudes of these four lifetime components are: 40 ps (41%), 130 ps (18%), 410 ps (17%) and 1.0 ns (24%), implying that the longest PSII related lifetime in fully open state is 1.0 ns (which is nearly five times as long as for WL-PSII). For the trace measured in closed state, the relative amplitudes of the four lifetime components are: 47 ps (36% amplitude), 180 ps (24%), 990 ps (28%) and 2.0 ns (12%): the longest PSII-related lifetime rises therefore to 2 ns. The average lifetime of FRL-CF cells at 735 nm (excluding the fastest component, which is mostly due to energy transfer) rises from 560 ps in open state to 880 ps in closed state, implying Fm/Fo = 1.6.

An overlay of the experimental and fitted TRF traces can be found in Supplementary Figure 20. The power-dependency (A) was investigated once, while the data in (B,C) are representative of 3 technical replicas and are consistent with the corresponding data measured with the Streak Camera (Figure 4C-D).

A)

B) C)

22

0,0 0,5 1,0 1,5 2,0 2,5 3,00,0

0,2

0,4

0,6

0,8

1,0

Nor

mal

ized

fluo

resc

ence

Time (ns)

0.2 uW 0.5 uW 1 uW 50 uW 50 uW + DCMU IRF

CT FRL

740 760 780-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

DAS

λ (nm)

0.05 ns 0.14 ns 0.45 ns 1.5 ns

CT FRL - open PSII

740 760 780-1.0

-0.5

0.0

0.5

1.0

DAS

λ (nm)

0.05 ns 0.20 ns 0.63 ns 1.9 ns

CT FRL - closed PSII

Supplementary Figure 19. TRF measurements of intact cells of FRL-adapted CT. A) Normalized TRF traces detected at 740 nm upon 438 nm excitation of cells of FRL-adapted CT at increasing powers (at RT). The IRF trace is shown in grey. The emission wavelength was chosen to enhance PSII contribution. Measurements were performed with a TCSPC setup at 10 MHz repetition rate. All traces were recorded for 10 minutes or until reaching 4000 counts at peak maximum. The traces become longer-lived at increasing powers due to an increasing amount of closed PSII RCs. The traces are power-independent at excitation powers below 1 μW. Due to the spectral heterogeneity of FRL-adapted cells, fluorescence traces detected at three distinct wavelengths (740, 755 and 790 nm) were used to perform the following global exponential fit (B,C).

B,C) Global analysis of TRF data of FRL-CT cells in open state (1 μW, (B)) and in closed state (50 μW + 50 μM DCMU, (C)) upon 438 nm excitation. Four components were required for a satisfactory global fit under both conditions. In open state, the relative amplitudes of these four lifetime components are: 50 ps (31%), 140 ps (35%), 450 ps (17%) and 1.5 ns (17%), implying that the longest PSII related lifetime in fully open state is 1.5 ns (which is more than six times as long as for WL-PSII). For the trace measured at in closed state, the relative amplitudes of the four lifetime components are: 50 ps (38% amplitude), 200 ps (31%), 630 ps (7%) and 1.9 ns (24%): the longest PSII-related lifetime rises therefore to almost 2 ns. The average lifetime of FRL-CT cells at 740 nm (excluding the fastest component, which is mostly due to energy transfer) rises from 550 ps in open state to 900 ps in closed state, implying Fm/Fo = 1.6.

An overlay of the experimental and fitted TRF traces can be found in Supplementary Figure 20. The power-dependency (A) was investigated once, while the data in (B,C) are representative of 3 technical replicas and are consistent with the corresponding data measured with the Streak Camera (Supplementary Figure 9C-D).

A)

B) C)

23

0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 0.95

Fluo

resc

ence

(a.u

.)

Time (ns)


0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 0.95

CF FRL-cells open

0 1 2

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 0.95

0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 1.03

Fluo

resc

ence

(a.u

.)

Time (ns)


0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.03

CF FRL-cells closed

0 1 2

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.03

0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 1.01

Fluo

resc

ence

(a.u

.)

Time (ns)


0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.01

CT FRL-cells open

0 1 2

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.01

0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 1.11

Fluo

resc

ence

(a.u

.)

Time (ns)


0 1 2 3 4

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.11

CT FRL-cells closed

0 1 2

0,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

(a.u

.)

Time (ns)


χ2 = 1.11

Supplementary Figure 20. TRF measurements of intact cells of FRL-adapted strains. Overlay of raw and globally fitted TRF traces of cells of FRL-acclimated CF and CT (measured with a TCSPC setup upon 438 nm excitation). For each line (representing a different dataset), three experimental time-traces are shown in grey corresponding to three selected emission wavelengths (specified in the legends). For each dataset, all traces detected at different wavelengths are globally analyzed as explained in the methods section obtaining the fitted traces (shown in black) and the global χ2 shown at each line. The corresponding residuals (i.e. the difference between experimental and fitted traces) are shown in blue. The DAS obtained from globally analyzing these data can be found in Supplementary Figures 18 (for cells of FRL-CF) and 19 (for cells of FRL-CT). For each sample, the traces are representative of 3 technical replicas yielding very similar results, which are also in agreement with those measured with the Streak Camera (Figure 4C-D and Supplementary Figure 9C-D).

24

400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

Abso

rptio

n

λ (nm)

600 650 700 750 800 8500.0

0.2

0.4

0.6

0.8

1.0

Nor

m. f

uore

scen

ce

λ (nm)

400 440

Supplementary Figure 21. Absorption and emission spectra of Acaryochloris Marina cells. The absorption maximum in the Qy is at 710 nm. The emission spectra were obtained after 400 nm and 440 nm excitation and are normalized to the peak maximum (723 nm). Data are representative of 2 highly similar biological replicas.

A) B)

25

680 720 760 8000

40

80

120

160

DAS

λ (nm)

50 ps 740 ps

AM - low power

680 720 760 8000

400

800

1200

1600

DAS

λ (nm)

48 ps 1.4 ns

AM - closed PSII

0 500 1000 15000.0

0.2

0.4

0.6

0.8

1.0

Nor

m. F

luor

esce

nce

Time (ps)

low power closed PSII

Supplementary Figure 22. TRF measurements of intact cells of Acaryochloris Marina (AM). A,B) 2D color maps representing TRF data of AM intact cells excited at 400 nm at low power (5 μW, when most PSII RCs are open, left side) and with closed PSII (50 μW + 50 μM DCMU, right side). Measurements were performed at RT with a Streak Camera setup at a repetition rate of 250 kHz.

C,D) DAS from global analysis of TRF measurements of AM cells in (A,B). Two lifetime components were sufficient to fit the data in both conditions. The two components maintain very similar spectra and

A) B)

C) D)

E)

26

amplitudes under both conditions. However, the lifetime of the short-lived component (about 50 ps) is not influenced by the excitation pressure, while that of the other component increases from 0.74 ns to 1.4 ns when PSII RCs close. For this reason, the short-lived component (which is also redshifted) can be assigned mostly to PSI kinetics, whereas the longest component to PSII kinetics. Note that the spectral difference between PSI and PSII DAS is not as pronounced as found for CF and CT cells grown in FRL (Figures 4C and Supplementary Figure 9C) or even in WL (Figures 4A and Supplementary Figure 9A). This suggests that the extent of red forms in the Chl d-containing PSI of AM is lower than in other types of PSI (containing Chl a only or both Chl a and f).

E) Fluorescence time traces from AM cells (400 nm excitation) integrated over the 710-730 nm range at low powers (blue) and in closed state (red). The overlay highlights the difference between the fluorescence kinetics measured when PSII RCs are mostly open or closed in the nanosecond range.

Data are representative of 2 highly similar technical replicas.

27

0 1 2 3 40,0

0,2

0,4

0,6

0,8

1,0

Fluo

resc

ence

Time (ns)

0.2 uW 0.3 uW 0.5 uW 1 uW 2 uW 10 uW 50 uW 50 uW DCMU IRF

0,0 0,5 1,0 1,5 2,0

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 1.07

AM cells open

Fluo

resc

ence

(a.u

.)

Time (ns)


0 1 2 3 4 5 6

0,0

0,2

0,4

0,6

0,8

1,0

χ2 = 1.00

AM cells closed

Fluo

resc

ence

(a.u

.)

Time (ns)


Supplementary Figure 23. TRF measurements of intact cells of Acaryochloris Marina (AM).

A) Normalized fluorescence time traces detected at 722 nm upon 438 nm excitation of AM cells at increasing powers (at RT). Measurements were performed with a TCSPC setup at 10 MHz repetition rate. All traces were recorded for 10 minutes or until reaching 2000 counts at peak maximum. The traces become longer-lived at increasing powers due to an increasing amount of closed PSII RCs. The traces are power-independent only at excitation powers below 0.5 μW, implying that the cells must be measured at very low powers to be kept in fully open state. The IRF trace detected at 722 nm is shown in grey.

An exponential fit of the fluorescence trace of AM cells in open state (0.2 μW) requires two lifetime components: 64 ps (55% amplitude) and 440 ps (45% amplitude), implying that the longest PSII-related lifetime in fully open state is 440 ps (which is nearly twice as long as for PSII of WL-adapted CF and CT). For the trace measured in closed state (50 μW + 50 μM DCMU), three lifetimes were needed: 51 ps (48% amplitude), 640 ps (12%) and 2.3 ns (40%). In both conditions, the shortest lifetime component (50-60 ps) can be ascribed mostly to PSI trapping (see also Supplementary Figures 22C-D). The long-lived components stem, instead, from PSII. The average lifetime of AM cells at 722 nm rises from 230 ps in open state to 1.0 ns in closed state, implying Fm/Fo = 4.3 (values of up to 5 have been previously reported)6, which is comparable to that of WL-PSII in vivo. This finding is somehow surprising, since PSII of AM in open state has a clearly longer lifetime than WL-PSII (Figure 4A). The relatively high ΦPSII in AM is due to the longer average lifetime of closed PSII, implying that closed PSII is less “quenched” in AM than in WL-strains.

B,C) Overlay of the corresponding experimental and fitted TRF traces. The power dependency (A) was investigated once, while the traces in (B,C) are representative of 2 highly similar technical replicas.

A)

B) C)

28

Supplementary Figure 24. Purification of FRL-photosystems. Resulting bands from sucrose density gradient ultracentrifugation of thylakoids from FRL-adapted cells as described in the Materials and Methods section of the manuscript. The bands containing PSII dimers and PSI trimers are indicated by arrows. The purification was performed twice for each strain starting from different cultures, yielding very similar spectroscopic properties for corresponding bands.

% Chl a Chl a per monomer

% Chl f Chl f per monomer

% Chl d Chl d per monomer

FRL-PSII CF 89.4 31.3 9.4 3.3 1.2 0.4 FRL-PSI CF 94.6 85.1 5.4 4.9 - - FRL-PSI CT 93.6 84.2 6.4 5.8 - -

Supplementary Table 1. Pigment quantification in isolated photosystems. Pigments were extracted in pure methanol and their concentrations in the extract were determined by absorbance measurements. The absorption spectra were fitted using the spectra and extinction coefficients of the pure pigments in methanol reported in Li et al.7 to yield their relative amount (with a standard deviation ≤ 0.2% based on 3 technical replicas). The relative pigment content was then normalized to the total number of Chls per PSII/PSI monomer (35 for PSII monomer8 and 90 for PSI monomer9) to obtain absolute numbers. Due to the significant heterogeneity of the preparation, the pigment content of FRL-PSII particles from CT is not shown in this table.

Chl Subunit coupling (cm-1) time (ps) Chl Subunit coupling (cm-1) time (ps) 405 D1 -18.2265 3.88 409 D1' 0.3 14334.06

CF CT

PSII PSII PSI PSI

29

406 D1 - 410 D1' -0.297 14587.57 407 D1 14.1045 6.48 411 D1' 0.2685 17838.12 410 D1 2.9175 151.57 414 D1' 0.3015 14240.02 602 CP47 -0.1035 120009.44 604 CP47' 0.3495 10524.41 603 CP47 1.0725 1120.47 605 CP47' -0.027 1721076.01 604 CP47 -0.3645 9736.88 606 CP47' -0.8205 1912.45 605 CP47 -0.5565 4161.24 607 CP47' 0.2625 18728.26 606 CP47 1.323 736.91 608 CP47' 0.8175 1927.54 607 CP47 -0.81 1964.23 609 CP47' -0.402 7977.56 608 CP47 -2.2575 252.95 610 CP47' 0.0195 3277588.90 609 CP47 -2.328 237.98 611 CP47' -0.462 6051.38 610 CP47 -0.8535 1768.42 612 CP47' 0.3555 10174.27 611 CP47 -1.053 1161.80 613 CP47' -0.72 2491.72 612 CP47 0.636 3182.93 614 CP47' 0.9015 1587.49 613 CP47 0.201 32036.40 615 CP47' 0.5475 4307.69 614 CP47 -1.3365 721.91 616 CP47' -0.6705 2865.82 615 CP47 -1.4385 623.77 617 CP47' 1.419 640.95 616 CP47 0.3105 13358.10 618 CP47' 0.258 19317.51 617 CP47 -0.6225 3320.29 619 CP47' 0.8685 1711.92 501 CP43 1.9725 331.29 902 CP43' -0.096 141476.08 502 CP43 -2.3625 231.15 903 CP43' 0.1305 75102.90 503 CP43 -0.255 19899.50 904 CP43' -0.012 8759469.59 504 CP43 1.83 385.20 905 CP43' -0.093 151027.29 505 CP43 7.452 23.22 906 CP43' -0.0435 685006.90 506 CP43 0.5265 4660.34 907 CP43' 0.0735 243313.89 507 CP43 2.6445 184.35 908 CP43' -0.1095 108644.43 508 CP43 -0.7635 2210.48 909 CP43' -0.0705 264480.54 509 CP43 -1.569 523.70 910 CP43' 0.105 116851.22 510 CP43 2.1285 284.60 911 CP43' -0.1185 92714.03 511 CP43 0.276 16976.01 912 CP43' -0.0735 235543.52 512 CP43 -0.3375 11282.97 913 CP43' -0.042 726671.46 513 CP43 0.7605 2228.23 914 CP43' -0.06 354580.64 402 D2 -90.8955 0.16 402 D2' -0.255 19808.97 403 D2 -0.0555 414749.91 403 D2' 0.1455 60933.28

Supplementary Table 2. Electronic couplings and hopping rates between the primary electron donor ChlD1 and all other Chls in PSII core. Calculations were based on the 1.9Å-resolution crystal structure of PSII core of Thermosynechococcus vulcanus (PDB: 3WU2)8. Electronic couplings with ChlD1 (in cm-1) were calculated in the point dipole approximation placing each transition dipole at the location of the central Mg and setting the Qy dipole strength of Chl f as 1.5 times that of Chl a (13.96 Debye2). The latter choice is justified by experimental data on the oscillator strengths of Qy transitions7. Both coupled Chls were assumed to be Chls f. Energy transfer lifetimes (in ps) were calculated in the Förster approximation, assuming that

30

the acceptor (ChlD1 in D1) absorbs at 727 nm and the donor (any other Chl) emits at 740 nm (to account for the red-most Chl f in PSII). The spectral shapes were assumed to be Gaussians with 𝜎𝜎 = 100 cm-1, which is reasonable at RT. Increasing the 𝜎𝜎 to 200 cm-1 or changing the spectral shape to a Lorentzian with a FWHM of 100-200 cm-1 (to account for a possibly different spectral broadening) did not change the rates drastically (the relative increase in the rates was no more than 50%). The couplings are also not very sensitive if the energy gap between donor and acceptor is below 20 nm, which is in the order of the chlorophyll spectral width. This implies that, at RT, the main factor limiting energy transfer between relatively isolated Chls f is their electronic coupling. The table displays corresponding Chls in the dimer side by side. The Chls in the antenna (CP43/47) resulting in hopping times shorter than 1 ns (roughly corresponding to coupling >1 cm-

1 with the primary donor) are highlighted in bold. Most of these Chls display weak electronic couplings with ChlD1 (absolute value < 3 cm-1), which result into rather slow hopping times (τ > 100 ps). If occupied by a Chl f, these sites represent possible kinetic bottlenecks in the energy equilibration towards the RC.

References 1. Ho, M.-Y. et al. Extensive remodeling of the photosynthetic apparatus alters energy transfer

among photosynthetic complexes when cyanobacteria acclimate to far-red light. Biochim. Biophys. Acta - Bioenerg. 148064 (2019). doi:10.1016/j.bbabio.2019.148064

2. Nürnberg, D. J. et al. Photochemistry beyond the red limit in chlorophyll f-containing photosystems. Science 360, 1210–1213 (2018).

3. Vassiliev, S., Lee, C. I., Brudvig, G. W. & Bruce, D. Structure-based kinetic modeling of excited-state transfer and trapping in histidine-tagged photosystem II core complexes from Synechocystis. Biochemistry 41, 12236–12243 (2002).

4. Miloslavina, Y. et al. Charge separation kinetics in intact photosystem II core particles is trap-limited. A picosecond fluorescence study. Biochemistry 45, 2436–2442 (2006).

5. Schmitt, F. J. et al. Photosynthesis supported by a chlorophyll f-dependent, entropy-driven uphill energy transfer in Halomicronema hongdechloris cells adapted to far-red light. Photosynth. Res. 139, 185–201 (2019).

6. Schiller, H., Senger, H., Miyashita, H., Miyachi, S. & Dau, H. Light-harvesting in Acaryochloris marina - Spectroscopic characterization of a chlorophyll d-dominated photosynthetic antenna system. FEBS Lett. 410, 433–436 (1997).

7. Li, Y., Scales, N., Blankenship, R. E., Willows, R. D. & Chen, M. Extinction coefficient for red-shifted chlorophylls: Chlorophyll d and chlorophyll f. Biochim. Biophys. Acta - Bioenerg. 1817, 1292–1298 (2012).

8. Umena, Y., Kawakami, K., Shen, J. R. & Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9Å. Nature 473, 55–60 (2011).

9. Kato, K. et al. Structural basis for the adaptation and function of chlorophyll f in photosystem I. Nat. Commun. 11, 238 (2020).

far-red absorption and light-use efficiency trade-offs in ...10.1038... · far-red absorption and...

Documents