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,*
1
Department 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.
5K
10 K
25 K
35 K
50 K
70 K
A
F695
B
1
5K
25 K
50 K
75 K
CP47
0
F689
Normalized Fluorescence (a.u.)
1
5K
10 K
25 K
35 K
50 K
75 K
CP43
0
1
C
0
670
680
690
700
710
Wavelength (nm)
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 PQA 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 Qy nonresonant persistent holes have a
profile similar to that obtained due to formation (in the presence of dithionite) of stable Pheo D1
(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 Pheo D1 in PSIIcc 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.
684
686
541.8
-2
b'
535
540
545
c
a
-0.02
650
0.4
693
0
685.5
Δ Absorbance
0
Absorbance
b
a'
2
Absorbance (a.u.)
0.02
Δ Absorbance 10  3
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.
0
660
670
680
690
700
Wavelength (nm)
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
PSII-cc1
PSII-cc2
PSII-cc3
PSII-cc4
Organism
Detergent
Isolation
Conditions
Oligomeric
State
Chl/P680
Pheo/P680
Chl b
-carotene
Cyt b559
QA/P680
QB/P680
Mn/P860
T. elongatus
β-DM
Detergent
extraction, tentacle
ion exchange
chromatography
PSII-cc5
T. elongatus
β-DM
Detergent
extraction,
centrifugation at
50,000 rpm
T. vulcanus
β-DM, LDAO
Detergent
extraction, anion
exchange
chromatography
Synechocystis 6803
β-DM
Detergent
extraction, anion
exchange
chromatography
3X crystallization
Spinach
β-DM
Detergent
extraction,
centrifugation at
17,000g, perfusion
chromatography
No crystallization
No crystallization
No crystallization
No crystallization
Dimer
Dimer
Dimer
Dimer
Dimer
N/R
32  1
34  2.1
37.50  2
2
2
N/R
2
N/R
N/R
N/R
0.8  0.2
N/R
71
8.6  1
9.50  0.5
N/R
N/R
N/R
1.16  0.1
N/R
N/R
1.08  0.1
2.9  0.8
N/R
N/R
1.08  0.1
N/R
4.5  0.5
3.6  0.7
3.92  0.2
2X (D1, D2, CP47, 2X (D1, D2, CP47, 2X (D1, D2 CP47,
2X (D1, D2 CP47,
CP43, CP29 (< 10
CP43, PsbE, F, H,
CP43, PsbE, F, I,
CP43, PsbE, F,
Protein
mol %), PsbE , F,
I, J, K, L, M, O, T,
K, L, T, Cyt c550,
several small
Subunits
O, W, several small
U, V, X, Y, Z)
several small
subunits)
subunits)
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)
37
2
0
N/R
N/R
1
0.8
N/R
2X (D1, D2, CP47,
CP43, PsbE, F, H,
I, J, K, L, M, O, T,
U, V, X, Y, Z)
Table 2. Structural/biochemical information and isolation conditions of various isolated RCs and PSII-m
Organism
Isolated RC1
Isolated RC1
PSII-m3
PSII-m4
Detergent
Isolation
Conditions
Spinach
β-DM, Triton X-100,
Detergent extraction from
PSII-enriched membranes,
differential centrifugation
at 100,000g, DEAE ionic
exchange chromatography,
Triton X-100 exchange
with β-DM, NaCl gradient
C. reinhardtii
β-DM, Triton X-100
Detergent extraction from
thylakoid membranes,
differential centrifugation
at 150,000g, DEAE ionic
exchange chromatography,
Triton X-100 exchange
with β-DM, NaCl gradient
Spinach
Triton X-100
Detergent extraction
Oligomeric
Isolated
Isolated
Supercomplex
State
5.85
5.7
Chl/P680
134  2
2
2
2
Pheo/P680
0
0
Chl b
62  1
N/R
N/R
91
-carotene
N/R
N/R
Cyt b559
1.0  0.2
Lost
Partly present
N/R
QA/P680
Lost
Lost
N/R
QB/P680
Destroyed
Destroyed
~5
Mn/P680
Protein
D1, D2, PsbE, F
D1, D2, PsbE, F
Complete Membranes
Subunits
N/R = not reported. 1Acharya et al. (2012a); 3Smith et al. (2002); 4Wang et al. (2002)
C. reinhardtii
Triton X-100
Detergent extraction,
centrifugation at 40,000g
Supercomplex
N/R
N/R
N/R
N/R
N/R
N/R
N/R
N/R
Complete Membranes
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