Supporting Information

Critical Assessment of the Emission Spectra of Various Photosystem II Core Complexes

Jinhai Chen,1 Adam Kell,1 Khem Acharya,1 Christopher Kupitz,2 Petra Fromme,2 and Ryszard Jankowiak1,3,*

1Department of Chemistry and 3Department of Physics, Kansas State University, Manhattan, Kansas 66506, USA; 2Department of Chemistry and Biochemistry, Arizona State University,

Tempe, Arizona 85287, USA

*Corresponding author; e-mail address: ryszard@ksu.edu

Temperature dependent emission spectra. Fig. 1 shows the temperature-dependent emission spectra of the Photosystem II core complex (PSII-cc) (Krausz et al. 2005a), intact CP43 (Dang et al. 2008; Reppert et al. 2008), and partly bleached CP47 (Acharya et al. 2010) (frames A-C, respectively). It appears that the F689 emission band (see the double arrow) observed in frames A and C is the same as the F695mod band assigned previously to the sub-population of CP47 complexes with “modified” low-energy states (Neupane et al. 2010), since annealing experiments largely recover the contribution from the lowest energy state, i.e., the F695 emission band (compare the 70 and 75 K spectra in frames A and C, respectively, with shapes of curves f and g in Fig. 1B of the main manuscript). A very weak contribution from 685 nm emission (F685 band; see short black arrow) most likely originates from a minor contribution from the destabilized CP47 complexes. It is possible that some pigments were photooxidized.

C

0

1 5 K10 K25 K35 K50 K70 K

0

15 K25 K50 K75 K

670 680 690 700 7100

1 5 K10 K25 K35 K50 K75 K

CP4

7

CP43

Nor

mal

ized

Flu

ores

cenc

e (a

.u.)

Wavelength (nm)

F695

B

A

F689

Fig. 1 Frame A shows the temperature dependent emission spectra of PSII-cc (Krausz et al. 2005a). Frames B and C show temperature-dependent emission spectra of intact CP43 (Dang et al. 2008; Reppert et al. 2008) and CP47 (Acharya et al. 2010), respectively. The CP47 spectra were measured after the sample was illuminated with 496.5 nm laser light (f ~ 4000 J/cm2) at 5 K

Hole-burned spectra. We briefly re-examine the (nonresonant) persistent saturated holes obtained for isolated reaction centers (RCs) (i.e., the D1/D2/Cyt b559 complex) from C. reinhardtii and spinach (Acharya et al. 2012a). The broad persistent holes in Fig. 2 (B = 665.0 nm) appear as a result of downhill energy transfer. Both holes revealed responses in the Qx region of the active pheophytin (PheoD1) near 545 1 (for C. reinhardtii, frame A) and 544 1 nm (for spinach, frame B), respectively (Acharya et al. 2012a). This suggests that the weak, narrow (fwhm of ~120 cm-1), nonresonant bleach is strongly contributed to by PheoD1. In isolated RCs the Qy transition of PhoeoD1, depending on sample quality, lies near 681-684 nm (see dashed arrows); similar Qy transitions of PhoeoD1 were obtained for 496.5 nm excitation (Acharya et al. 2012a; Jankowiak et al. 1999, 2002; Konermann and Holzwarth 1996; Krausz et al. 2008; Mimuro et al. 1995; Stewart et al. 2000; Vasil’ev et al. 2001), though the vertical energies of PheoD1 in PSII-cc were also placed near 666 nm (Lewis et al. 2013; Romero et al. 2014; Shibata et al. 2013). Note that site energies can be directly compared only if the same reorganization energy is used in theoretical calculations; that is, vertical and (0,0) transition energies cannot be directly compared. There is even less agreement regarding the site energy of PheoD2 (Jankowiak et al. 1999, 2002; Konermann and Holzwarth 1996; Lewis et al. 2013; Mimuro et al. 1995; Romero et al. 2014; Shibata et al. 2013; Stewart et al. 2000; Vasil’ev et al. 2001).

Fig. 2 Nonresonant, persistent NPHB spectra obtained for RCs from isolated C. reinhardtii (frame A) and spinach (frame B) (Acharya et al. 2012a). Both spectra revealed bleaching in the Qx absorption band of PheoD1 near 545 and 544 nm, respectively, whose position depends on sample intactness. Both spectra were obtained with λB = 665.0 nm and measured at 5 K

Note that in the case of a persistent nonphotochemical hole burned (NPHB) spectrum, there is no electrochromic shift and there are no charges on chlorophyllD1 (ChlD1) (at least in isolated spinach RC without QA) that could lead to the electrochromic shift. We suggest that bleaching of PheoD1 can occur during the long-lived triplet state, i.e., 3ChlD1. A small PheoD1 shift was observed for isolated RCs from C. reinhardtii (Acharya et al. 2012a) since in this preparation a small subpopulation of RCs contained QA, and as a result, these RCs could form the PQA

state. Although hole shapes in Fig. 2 are similar, the broad hole in C. reinhardtii, located at 683.8 nm, is about 3.2 nm red-shifted in comparison with the hole typically obtained for RCs isolated from spinach (Acharya et al. 2012a; Chauvet et al. 2015). The Qy and Qx

spectral positions of the latter hole in isolated RCs from spinach also varied from preparation to preparation (Acharya et al. 2012a; Chauvet et al. 2015). We hasten to add that the Q y

nonresonant persistent holes have a profile similar to that obtained due to formation (in the presence of dithionite) of stable PheoD1

(Jankowiak et al. 1999), further supporting the assignment that PheoD1 may contribute near the 680-684 nm spectral region. Very recently, Acharya et al. (2012a, 2012b) and Chauvet et al. (2015) proposed that the RC from C.

reinhardtii is more intact than that isolated from spinach and the persistent hole near 684 nm, even in isolated RCs, can be assigned as bleaching of PheoD1 (see frame A of Fig. 2).

We want to mention here that so far charge-transfer (CT) emission has not been observed in isolated RCs from C. reinhardtii nor spinach, and, in general, the isolated RC samples are heterogeneous mixtures of intact and destabilized D1/D2/Cyt b559 complexes (Acharya et al. 2012a, 2012b; Chauvet et al. 2015). Thus, it is very likely that the site energy of PheoD1 in PSII-cc near 685 nm (as proposed by Krausz et al. (2005a) and Masters et al. (2001)) may not only be correct, but the 686-687 nm emission could originate from the pigments in closed RCs, e.g., from Chls and Pheos contributing to the lowest excitonic state of the RCs, and, at least in part, from the transiently decoupled PheoD1 (vide infra) during the long lived 3P state.

Fig. 3 shows the 5 K absorption spectrum of Thermosynechococcus (T.) elongatus PSII-cc and the corresponding persistent (curve b) and transient (curve c) HB spectra (B = 496.5 nm). The bleach near 693 nm in spectrum b corresponds to the lowest energy band of CP47, in agreement with our data reported for the isolated (intact) CP47 complex (Acharya et al. 2010; Neupane et al. 2010; Reppert et al. 2010). We suggest that the major band near 685.5 nm corresponds to the lowest energy state in the closed RCs, though a contribution from the peripheral antennas (CP43 and/or CP47) cannot be entirely excluded. However, comparison to previous nonresonant HB spectra for spinach PSII-cc (Reppert et al. 2010) reveals more bleach at ~685 nm for PSII-cc from T. elongatus. The ΔΔA spectrum, i.e., ΔHB, of Reppert et al. (2010) (see top inset of Figure 2 therein) was found to be contributed to solely by the nonresonant HB spectra of intact, isolated CP43 and CP47 complexes. Theoretical description of the holes presented above is beyond the scope of this manuscript as there is no agreement in the literature regarding the site energies of various antenna and RC pigments (Müh et al. 2012; Raszewski and Renger 2008; Reppert et al. 2008, 2010; Romero et al. 2014; Shibata et al. 2013). Here it suffices to say that the remaining negative and positive bands in spectrum b at higher energies are due to the bleaching of the pigment(s) contributing to the lowest energy state(s) mentioned above, which leads to modified excitonic interactions.

650 660 670 680 690 700-0.02

0

0.02

0

0.4

Wavelength (nm)

ΔA

bsor

banc

eA

bsorbance

693

684

Δ A

bsor

banc

e

10

368

6

Absorbance (

a.u.)

-2

2

535 540 545

0 541.

8

685.

5

a

b

c

a'

b'

Fig. 3 Low-temperature (5K) spectra obtained for PSII-cc of T. elongatus (closed RC). Curve a is the absorption spectrum. Spectra b and c are the persistent (saturated) and transient holes, respectively, obtained with B = 496.5 nm. The inset shows the Qx absorption region of Pheos (curve a') and the Qx bleach (curve b') corresponding to the persistent hole, i.e., spectrum b. The

black dotted curve in the inset (superimposed on curve b') is the fit to several holes burned in different experiments

Note that spectrum c (i.e., the transient hole) was obtained after saturation of the persistent hole (spectrum b) and, as a result, reveals mostly the new (modified) lowest energy state near 686 nm. This state (at 5 K) most likely corresponds to the lowest energy state of the RC pigments (revealed via the triplet state(s), i.e., 3P or 3ChlD1) and/or a triplet of photo-bleached CP47. This is consistent with the fact that curve c does not possess any bleach in the Qx region of Pheos. In contrast, curve b has bleach near 541.8 nm (see curve b' in the inset); as expected the bleach in the Qx region of PheoD1 is blue-shifted to about 542 nm due to the electrochromic shift observed in the closed RC within the PSII-cc, in agreement with the data of Krausz et al. (2005b). Subsequent burning with B = 496.5 nm led to a persistent hole which clearly revealed the Qx transition of electrochromically shifted PheoD1 (see inset).

The question arises as to what is the origin of the broad nonresonant ~680, ~684, and ~685 nm persistent holes (in Figures 2 and 3), which are accompanied by a bleach in the Qx

region of PheoD1? For isolated RCs, NPHB can occur during the long-lived triplet or CT states (Acharya et al. 2012a). Bleaching during the charge separated states is only possible if QA is present, otherwise fast recombination leads to 3ChlD1. Thus, while the continuous wave laser is on, it may be possible to bleach the temporarily decoupled PheoD1. A similar argument can be made for PSII-cc and membrane PSII (PSII-m) (with intact RCs and QA present). While illuminated PSII-cc most likely contains closed RCs (i.e., with QA

–), with only a fraction of P680 open. If a closed RC is reduced, further charge separation may be possible (i.e., ChlD1

+PheoD1–),

which would quickly recombine and form 3ChlD1. The temporarily decoupled PheoD1 could then act as an energy trap for energy transfer from higher energy states (being excited during illumination).

Sample Isolation and Preparation. Tables 1 and 2 provide structural/biochemical information and isolation conditions of various PSII-cc and RC/PSII-m, respectively.

Table 1. Structural/biochemical information and isolation conditions of various PSII-cc

Organism

PSII-cc1

T. elongatus

PSII-cc2

Spinach

PSII-cc3

T. elongatus

PSII-cc4

T. vulcanus

PSII-cc5

Synechocystis 6803Detergent β-DM β-DM β-DM β-DM, LDAO β-DM

Isolation Conditions

Detergent extraction, tentacle

ion exchange chromatography

3X crystallization

Detergent extraction,

centrifugation at 17,000g, perfusion chromatography

No crystallization

Detergent extraction,

centrifugation at 50,000 rpm

No crystallization

Detergent extraction, anion

exchange chromatography

No crystallization

Detergent extraction, anion

exchange chromatography

No crystallizationOligomeric

State Dimer Dimer Dimer Dimer Dimer

Chl/P680 37 32 1 34 2.1 N/R 37.50 2Pheo/P680 2 2 2 N/R 2

Chl b 0 0.8 0.2 N/R N/R N/R-carotene N/R 7 1 8.6 1 N/R 9.50 0.5

Cyt b559 N/R N/R N/R N/R 1.16 0.1QA/P680 1 N/R 2.9 0.8 N/R 1.08 0.1QB/P680 0.8 N/R N/R 1.08 0.1Mn/P860 N/R 4.5 0.5 3.6 0.7 N/R 3.92 0.2

Protein Subunits

2X (D1, D2, CP47, CP43, PsbE, F, H, I, J, K, L, M, O, T,

U, V, X, Y, Z)

2X (D1, D2, CP47, CP43, CP29 (< 10 mol %), PsbE , F,

O, W, several small subunits)

2X (D1, D2, CP47, CP43, PsbE, F, H, I, J, K, L, M, O, T,

U, V, X, Y, Z)

2X (D1, D2 CP47, CP43, PsbE, F, I, K, L, T, Cyt c550,

several small subunits)

2X (D1, D2 CP47, CP43, PsbE, F, several small

subunits)

N/R = not reported. 1Kupitz et al. (2014a, b); 2Smith et al. (2002); 3Kern et al. (2005); 4Shen and Kamiya (2000); 5Tang and Diner (1994)

Table 2. Structural/biochemical information and isolation conditions of various isolated RCs and PSII-mOrganism Isolated RC1

Spinach

Isolated RC1

C. reinhardtii

PSII-m3

Spinach

PSII-m4

C. reinhardtiiDetergent β-DM, Triton X-100, β-DM, Triton X-100 Triton X-100 Triton X-100Isolation

ConditionsDetergent extraction from PSII-enriched membranes, differential centrifugation at 100,000g, DEAE ionic

exchange chromatography, Triton X-100 exchange

with β-DM, NaCl gradient

Detergent extraction from thylakoid membranes,

differential centrifugation at 150,000g, DEAE ionic

exchange chromatography, Triton X-100 exchange

with β-DM, NaCl gradient

Detergent extraction Detergent extraction, centrifugation at 40,000g

Oligomeric State Isolated Isolated Supercomplex Supercomplex

Chl/P680 5.85 5.7 134 2 N/RPheo/P680 2 2 2 N/R

Chl b 0 0 62 1 N/R-carotene N/R N/R 9 1 N/R

Cyt b559 N/R N/R 1.0 0.2 N/RQA/P680 Lost Partly present N/R N/RQB/P680 Lost Lost N/R N/RMn/P680 Destroyed Destroyed ~5 N/RProtein

Subunits D1, D2, PsbE, F D1, D2, PsbE, F Complete Membranes Complete Membranes

N/R = not reported. 1Acharya et al. (2012a); 3Smith et al. (2002); 4Wang et al. (2002)

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