Vol 464 | 22 April 2010 | doi:10.

1038/nature08885

LETTERS
Isolation of the elusive supercomplex that drives
cyclic electron flow in photosynthesis
Masakazu Iwai1*{, Kenji Takizawa1*, Ryutaro Tokutsu1, Akira Okamuro2, Yuichiro Takahashi2 & Jun Minagawa1

Photosynthetic light reactions establish electron flow in the mode of electron flow (Fig. 1a). Under weak light (20 mmol photons
chloroplast’s thylakoid membranes, leading to the production of m22 s21), we induced State 1 or State 2 by adding a PSII inhibitor,
the ATP and NADPH that participate in carbon fixation. Two 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), to oxidise the
modes of electron flow exist—linear electron flow (LEF) from PQ pool, or an uncoupler, carbonyl cyanide p-trifluoromethoxyphe-
water to NADP1 via photosystem (PS) II and PSI in series1 and nylhydrazone (FCCP), to reduce the PQ pool9, respectively. The
cyclic electron flow (CEF) around PSI (ref. 2). Although CEF is change of relative antenna size between PSI and PSII, assayed by
essential for satisfying the varying demand for ATP, the exact low temperature fluorescence emission spectra, confirmed the state
molecule(s) and operational site are as yet unclear. In the green inductions (Fig. 1b). We examined CEF activity in those cells by
alga Chlamydomonas reinhardtii, the electron flow shifts from measuring the P700 oxidation state in the presence of DCMU5.
LEF to CEF on preferential excitation of PSII (ref. 3), which is Upon actinic illumination, most of the P700 in the State 1 cells was
brought about by an energy balancing mechanism between PSII oxidised because LEF was inhibited by DCMU and CEF was largely
and PSI (state transitions4). Here, we isolated a protein super- suppressed; about half of the P700 in the State 2 cells was rapidly
complex composed of PSI with its own light-harvesting complex re-reduced by the increased CEF (Fig. 1c). The increased CEF was
(LHCI), the PSII light-harvesting complex (LHCII), the cytochrome
b6f complex (Cyt bf), ferredoxin (Fd)-NADPH oxidoreductase a Linear electron flow Cyclic electron flow
(FNR), and the integral membrane protein PGRL1 (ref. 5) from
C. reinhardtii cells under PSII-favouring conditions. Spectro-
DCMU
scopic analyses indicated that on illumination, reducing equivalents
from downstream of PSI were transferred to Cyt bf, whereas oxi- DBMIB P700 DBMIB P700
dised PSI was re-reduced by reducing equivalents from Cyt bf, indi-
cating that this supercomplex is engaged in CEF (Supplementary PSII Cyt bf PSI Cyt bf PSI
Fig. 1). Thus, formation and dissociation of the PSI–LHCI–LHCII– CEF supercomplex
FNR–Cyt bf–PGRL1 supercomplex not only controlled the energy
balance of the two photosystems, but also switched the mode of b c State 2 + DBMIB

photosynthetic electron flow.

Fluorescence

CEF is crucial for a proper balance of NADPH and ATP in the

thylakoid stroma of photosynthetic organisms6 and for protecting
their photosynthetic apparatus from photodamage7. Two possible State 1
P700 oxidation

electron transfer pathways have been proposed for CEF—an Fd- State 2 + AA
dependent pathway and an NAD(P)H dehydrogenase (NDH)-
dependent pathway7. In the NDH-dependent pathway, NDH mediates
NADPH oxidation and plastoquinone (PQ) pool reduction much as 600 700 800
complex I does in mitochondria8. How electrons on Fd are transferred Wavelength (nm)
State 2
to the PQ pool in the Fd-dependent CEF (Fd-CEF), however, is still
unclear.
It has been reported that in C. reinhardtii, photosynthetic electron
flow switches between LEF and CEF depending on the excitation
100 ms
balance of the two photosystems, which is controlled by a photoac-
climation mechanism called state transition4. On preferential excita- Dark Light
tion of PSII, LHCII migrates to PSI, thereby redistributing excitation
energy from PSII to PSI (State 2, where CEF is predominant), and on Figure 1 | State 2 transition induction of CEF. a, LEF and CEF are
preferential excitation of PSI, LHCII migrates back to PSII, thereby schematically shown with their inhibitor binding sites. b, 77 K fluorescence
emission spectra in State 1 (blue) and State 2 (red) cells. The excitation
redistributing excitation energy from PSI to PSII (State 1, where LEF wavelength was 440 nm, and the emission spectra were normalized to a
is predominant)3. Thus, it should be possible to locate a functional shoulder at 699 nm. c, In vivo analysis of P700 oxidation estimated from
CEF unit in State 2 C. reinhardtii cells. DA810. State 1 and State 2 cells were exposed to actinic illumination by white
In this study, we first tested whether, under our experimental light-emitting diode (LED; 4,400 mmol photons m22 s21) with 10 mM
conditions, wild-type C. reinhardtii cell states were related to the DCMU and additional inhibitors (20 mM AA, 40 mM DBMIB), as indicated.
1
Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan. 2Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushima-naka,
Okayama 700-8530, Japan. {Present address: Real-Time Bio-Imaging Research Team, Extreme Photonics Research Group, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako,
Saitama 351-0198, Japan.
*These authors contributed equally to this work.

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NATURE | Vol 464 | 22 April 2010 LETTERS

sensitive to dibromothymoquinone (DBMIB), a Qo site inhibitor of was determined to be 0.97 6 0.03 from the redox difference spectra of
Cyt bf, but insensitive to antimycin A (AA). A4 (Fig. 2b). It is noteworthy that PGR5, a previously proposed
We previously isolated the PSI–LHCI–LHCII supercomplex by essential component for Fd-CEF in Arabidopsis thaliana12, was not
solubilising State 2 cell thylakoid membranes with the detergent present in any of the fractions, including A4, which was determined
n-dodecyl-b-D-maltoside10. In this study, we solubilized them with by both immunoblotting (Supplementary Fig. 3) and MS/MS ana-
n-tridecyl-b-D-maltoside (TM), a detergent with a one-unit longer lysis (Supplementary Table 1). This is probably because it was only
aliphatic chain that has been used successfully to purify the PSII– expressed at a very low level in C. reinhardtii. Alternatively, it might
LHCII supercomplex11, and we loaded the solubilized membranes have been lost during the biochemical manipulations as it is a small
onto a sucrose density gradient (SDG). Four distinct green bands protein without a transmembrane region peripherally associated
were separated (A1, A2, A3 and A4), with significantly more of the with the thylakoids12.
A4 fraction observed in State 2 than in State 1 (Supplementary Fig. 2). We used four different methods to check whether the proteins in
The protein composition of each green band was determined by the A4 fraction were forming a single supercomplex. (1) We recorded
SDS–polyacrylamide gel electrophoresis (SDS–PAGE), immuno- a molecular mass chromatogram during gel-filtration. Only a single
blots and MS/MS (tandem mass spectrometry) analysis. A1 con- peak was obtained for A4, with a molecular mass corresponding to
tained free LHCII proteins, including major trimeric LHCII types 1,400–1,600 kDa (Supplementary Fig. 4). (2) When we partially dis-
I–IV and minor monomeric LHCII CP26 and CP29; A2 contained assembled the A4 fraction complex(es) by NaCl treatment, the A4
the PSII core complex, A3 contained the PSI–LHCI supercomplex, band disappeared, and several bands with smaller molecular masses
and A4 contained the PSI–LHCI supercomplex, major trimeric emerged (Supplementary Fig. 5)—LHCII and PGRL1 in A1, Cyt bf in
LHCII types I–IV, minor monomeric LHCII (CP26 and CP29), A2, and the PSI–LHCI supercomplex in A3, indicating that they were
Cyt bf (Cyt b6, Cyt f, PETC, PetD and PETO), FNR, and PGRL1 originally forming a (super-)supercomplex. (3) We tested A4 band
(Fig. 2a, Supplementary Fig. 3 and Supplementary Table 1). Each formation in mutants deficient in a redox component in the A4
polypeptide in the A4 fraction except CP26 seemed to be at a near fraction. When thylakoids from PSI- or Cyt bf-deficient mutants
stoichiometric amount (Fig. 2a). The more precise Cyt bf/PSI ratio were solubilized and loaded onto an SDG, the A4 band was absent
in both cases (Fig. 2c), which indicates that both PSI and Cyt bf were
a (kDa) b necessary for formation of the A4 supercomplex. (4) We purified the
66 P700 A4 supercomplex from a His-tagged psaA mutant13 and subjected the
PsaA/B solubilized thylakoids to nickel affinity column chromatography.
Cyt b
Cyt f
Immunoblot analysis of the eluates showed the same protein list as
ΔARED-OX

Cyt f the original A4 fraction, including the PSI–LHCI supercomplex,

44 PGRL1 Dithionite major and minor LHCII, Cyt bf, FNR and PGRL1 (Fig. 2d).
FNR Neither PGR5 nor Fd was detected in the affinity-purified A4 super-
CP26
CP29 complex (Fig. 2d). Taken together, these observations indicate that
LhcbM5 Ascorbate
the proteins in the A4 fraction formed a single supercomplex.
30 a4
a6 But was the A4 supercomplex a functional unit for CEF? To
a3 ΔA=0.005
a2
address this question directly, we examined the electron transfer
20 a5
a7/PETC 500 600 700 activities of the supercomplex at its stromal and luminal sides by a
a8 Wavelength (nm) series of spectroscopic analyses. We evaluated the stromal side elec-
Cyt b6
PSAD
tron transfer by Cyt b reduction14,15. Cyt bf contains two b-type
a1/a9
d 1 2 3
haems, bH and bL (Fig. 3a). When Cyt b in the A4 supercomplex
PSAF D1 was reduced by NADPH in the presence of Fd, it produced a signal
14 PSAE CP47 about half (0.48 6 0.13) as large as the signal produced when both
LhcbM5
b-type haems were reduced by dithionite (Fig. 3b). That indicated
that one of the b-type haems in Cyt bf, most likely the high-potential
CP26 haem bH as suggested in the literature15, was reduced by NADPH via
c WT ΔPSI ΔCyt bf CP29 Fd and the bound FNR. This half-reaction of the stromal side Fd-
CEF—the electron transfer from NADPH to haem bH—was not
PsaA
inhibited by AA (Fig. 3b inset), which is consistent with the in vivo
PSAF assay described above (Fig. 1c).
A1 Cyt b6 We further examined the complete reaction of stromal side Fd-
CEF, namely the photoreduction of Cyt b. When we illuminated the
A2 Cyt f
A4 supercomplex, Cyt b was photoreduced in the presence of Fd
A3 PETC (Fig. 3c). Notably, most of Cyt f (554 nm), which was reduced by
FNR ascorbate, remained reduced during the measurements (Fig. 3c
inset). We conclude that the A4 supercomplex can carry out the
PGRL1
A4 electron transfer from P700 to one of the b-type haems in Cyt bf,
PGR5 likely haem bH, with the help of soluble Fd.
Fd We evaluated luminal side electron transfer by studying the flash-
induced kinetics of P7001 re-reduction and Cyt f oxidation16 within
Figure 2 | Profiling the A4 supercomplex formed in State 2. a, SDS–PAGE the A4 supercomplex (Fig. 3d). After being oxidised by a single-
profile of the A4 fraction stained with Coomassie brilliant blue R 250. turnover actinic flash, most of the P700 in the A4 supercomplex
Labelled proteins were identified by immunoblot (Supplementary Fig. 3) was re-reduced by reduced Pc, confirming that most (< 80%) of
and MS/MS analyses (Supplementary Table 1). Labels a1–a9 represent
the P700 in the A4 supercomplex accepted electrons from Pc
Lhca1–Lhca9, respectively. b, Redox difference spectra of the A4 fraction.
c, SDG of State 2 thylakoids from wild-type (WT), PSI-less mutant (DPSI) (Fig. 3e). The fraction of active P700 was equivalent to that reported
and Cyt bf-less mutant (DCyt bf) cells. d, Polypeptide profile of the A4 for purified PSI (70–90%)16,17. In the PSI–LHCI supercomplex (A3),
supercomplex purified by Ni-affinity chromatography as shown by which does not contain Cyt bf, P7001 reduction with substoichio-
immunoblot analysis. Lane 1, thylakoid membranes; lane 2, A3 fraction; lane metric amounts of Pc (0.1 mM Pc and < 0.5 mM P7001) was biphasic,
3, affinity-purified A4 supercomplex. which can be accounted for by fast reduction with reduced Pc and
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©2010 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 464 | 22 April 2010

a b c

0.002
0.002
NADPH

ΔA

Cyt b reduction
ΔA
ΔA=0.002
–AA
+ Fd
+AA

ΔARED-OX
Fd
Dithionite 540 560 580
bH 560 580
Wavelength (nm)

bL NADPH + Fd
– Fd
Fd
f P700 2s
Cyt bf PSI NADPH Dark Light
540 560 580
Wavelength (nm)

d e f
No Pc
MV
FNR Pc 1 µM

Cyt f oxidation
P700 oxidation

bH
Pc 0.1 µM
bL Pc 1 µM
(A3 : Pc 0.1 µM)
f P700
No Pc
Cyt bf Pc PSI Pc 0.1 µM
Pc –0.2 0.0 0.2 0.4 –0.2 0.0 0.2 0.4
Time (s) Time (s)

Figure 3 | CEF activity in the A4 supercomplex. a, Schematic diagram of AA. c, Cyt b photoreduction with and without 1 mM Fd. Inset, absorbance
stroma side electron transfer. b, Cyt b reduction by 3 mM dithionite, 1 mM changes after 2.5 s illumination. d, Diagram of luminal side electron transfer.
NADPH or 1 mM Fd, as indicated, in the presence of 5 mM Na-ascorbate. MV, methyl viologen. e and f, Flash-induced P7001 re-reduction and Cyt f
Inset, Cyt b reduction by 1 mM NADPH 1 1 mM Fd with and without 20 mM oxidation, respectively.

slow reduction with re-reduced Pc by ascorbate (Fig. 3e, black line) as was able to identify or purify FQR. In the meantime, Cramer and his
suggested previously16,17. In the A4 supercomplex, however, only the co-workers reported that Cyt bf was co-purified with FNR and was
fast reduction phase was observed with a substoichiometric amount reduced by Fd15. The present study demonstrates that Fd-CEF can
of Pc, indicating that Pc was shuttled between Cyt f and P700 (Fig. 3e, operate in a supercomplex without any unidentified components such
green line). Finally, we confirmed photo-oxidation of Cyt f in the A4 as the suggested FQR23. We conclude that the components included in
supercomplex. Cyt f was rapidly oxidised when exposed to actinic the current supercomplex are the primary components for Fd-CEF
light only in the presence of Pc (Fig. 3f). The kinetics (that is, half functionality.
time) were similar for Cyt f oxidation and P7001 reduction, indi- Besides its inherent advantage in CEF activity, super-supercomplex
cating that the overall reaction was limited by Pc binding to P700 formation may also enable CEF to operate independently of LEF.
rather than by Pc shuttling between Cyt f and P700. Because CEF and LEF share many redox carriers (for example, PQ,
Physical association of PSI, Cyt bf and FNR has long been sug- Cyt bf, Pc, PSI, Fd and FNR), they are potentially in competition with
gested as a platform for Fd-CEF18. Mathematical modelling of the one another. Furthermore, the redox poise of the CEF components
electron transfer19 and in vivo observation of its high efficiency18 could be disturbed if reduced LEF components coexist24. The super-
suggests that Fd-CEF operates in a supercomplex. More recently, complex formation compartmentalises CEF by localizing the mobile
physical interactions between PGRL1, which was essential for Fd- electron carriers (PQ, Fd and Pc), which would lead to the formation
CEF in A. thaliana, and PsaD (subunit of PSI), PetB (Cyt b6), FNR of a functional pool of CEF components.
and PGR5 were shown by a yeast two-hybrid assay using the corres- Above all, the CEF supercomplex allows us to witness the merger of
ponding genes from A. thaliana5. However, the enigmatic ‘super- two intensely studied physiological events in this field, state transi-
supercomplex’ comprising PSI, Cyt bf and FNR has never been tions and CEF, both of which were discovered more than four decades
detected and isolated5,20. The present study in C. reinhardtii, however, ago. Formation and dissociation of the CEF supercomplex is signifi-
demonstrates that when cells are in State 2, where more LHCIIs are cant not only in balancing the energy of the two photosystems, but also
associated with PSI, the CEF supercomplex is indeed present. in maintaining cellular ATP homeostasis by switching the electron
Moreover, the isolated supercomplex contained PGRL1 as well as a flow. An open question then arose: What is the molecular mechanism
loosely bound Cyt bf subunit PETO21 (Supplementary Table 1), but that controls formation of the CEF supercomplex? The tight coupling
did not contain PGR5. Notably, electron transfer activity was of state transitions and CEF in C. reinhardtii may provide a clue for
detected in this supercomplex on both the stromal and luminal sides. this problem as well. The association of LHCII and Cyt bf with PSI
The exact electron transfer pathway within Cyt bf is still unclear, but described here is indeed in line with their behaviours in the membrane
we speculate that the reducing equivalent on haem bH is transported domains during a transition from State 1 to 2. During that transition,
to the luminal side via PQ molecules in the thylakoid membranes LHCII and Cyt bf migrate from the appressed region in the thylakoid
(Supplementary Fig. 1). membranes, where PSII resides, to the non-appressed region, where
In early studies, Fd-CEF was characterized as AA-sensitive and was PSI resides25. Therefore, investigation on how state transitions and the
proposed to involve Cyt bf (ref. 22). Later studies did not confirm the protein relocations thereby are coupled with the membrane domain
AA-sensitivity of Cyt b reduction and led to the hypothesis that a function may shed new light on this problem. As has been reported in
unique AA-binding enzyme, Fd-PQ oxidoreductase (FQR), bypasses A. thaliana5, the PGRL1–PGR5 complex might interact with PSI and
Cyt bf and interfaces with PQ pool reduction by Fd23. No one, however, facilitate the formation of the CEF supercomplex also in C. reinhardtii
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NATURE | Vol 464 | 22 April 2010 LETTERS

if we assume that PGR5 was lost during the thylakoid preparations. 14. Lam, E. & Malkin, R. Ferredoxin-mediated reduction of cytochrome b-563 in a
chloroplast cytochrome b-563/f complex. FEBS Lett. 141, 98–101 (1982).
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15. Zhang, H., Whitelegge, J. P. & Cramer, W. A. Ferredoxin: NADP1 oxidoreductase
PETO was detected in the CEF supercomplex (Supplementary Table is a subunit of the chloroplast cytochrome b6f complex. J. Biol. Chem. 276,
1) may indicate another mechanism. Because this loosely-bound Cyt 38159–38165 (2001).
bf subunit is reversibly phosphorylated under State 2 conditions much 16. Rich, P. R., Heathcote, P. & Moss, D. A. Kinetic studies of electron transfer in a
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replaces the role of PGR5 in C. reinhardtii and acts as a state transition-
17. Hippler, M., Drepper, F., Farah, J. & Rochaix, J. D. Fast electron transfer from
inducible regulator for the CEF supercomplex. We are in favour of this cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii
hypothesis because it could account for (1) why the CEF induction requires PsaF. Biochemistry 36, 6343–6349 (1997).
depends on state transitions in C. reinhardtii3, (2) why not in A. 18. Joliot, P. & Joliot, A. Cyclic electron transfer in plant leaf. Proc. Natl Acad. Sci. USA
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19. Laisk, A. Mathematical modelling of free-pool and channelled electron transport
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METHODS SUMMARY 21. Hamel, P., Olive, J., Pierre, Y., Wollman, F.-A. & de Vitry, C. A new subunit of
cytochrome b6f complex undergoes reversible phosphorylation upon state
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20 mmol photons m22 s21 in Tris-acetate-phosphate media27 aerated with ambient 22. Tagawa, K., Tsujimoto, H. Y. & Arnon, D. I. Role of chloroplast ferredoxin in the
air at 23 uC. A mutant deficient in PSI, DPSI, was generated biolistically with a energy conversion process of photosynthesis. Proc. Natl Acad. Sci. USA 49,
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Low temperature fluorescence emission spectra10,11 and redox difference absorp- thylakoid membranes upon state transitions. Proc. Natl Acad. Sci. USA 88,
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cytochrome b6/f complex. I. Characterization of the complex subunits in
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Chlamydomonas reinhardtii. Biochim. Biophys. Acta 851, 229–238 (1986).
Published online 4 April 2010.
29. Avenson, T. J., Cruz, J. A. & Kramer, D. M. Modulation of energy-dependent
1. Arnon, D. I., Whatley, F. R. & Allen, M. B. Assimilatory power in photosynthesis: quenching of excitons in antennae of higher plants. Proc. Natl Acad. Sci. USA 101,
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cyclic electron flow in Chlamydomonas reinhardtii. EMBO Rep. 3, 280–285 (2002). Supplementary Information is linked to the online version of the paper at
4. Rochaix, J. D. Role of thylakoid protein kinases in photosynthetic acclimation. FEBS www.nature.com/nature.
Lett. 581, 2768–2775 (2007). Acknowledgements We thank S. Ozawa and M. Kuwano for technical help with
5. DalCorso, G. et al. A complex containing PGRL1 and PGR5 is involved in the switch MS/MS analysis, D. Kramer for valuable discussions, T. Shikanai, D. Leister and
between linear and cyclic electron flow in Arabidopsis. Cell 132, 273–285 (2008). T. Hase for providing antibodies, and K. Redding for providing the PSI-His mutant.
6. Kramer, D. M., Avenson, T. J. & Edwards, G. E. Dynamic flexibility in the light This study was supported by the Research Fellowship for Young Scientists from the
reactions of photosynthesis governed by both electron and proton transfer Japan Society for the Promotion of Science (M.I., R.T.), Grants-in-Aid for Scientific
reactions. Trends Plant Sci. 9, 349–357 (2004).
Research from the Ministry of Education, Culture, Sports, Science and Technology
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International Cooperative Program by Japan Science and Technology Agency
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9. Bulté, L., Gans, P., Rebeille, F. & Wollman, F. A. ATP control on state transitions in Author Contributions M.I. and R.T. purified supercomplexes and Pc, and
vivo in Chlamydomonas reinhardtii. Biochim. Biophys. Acta 1020, 72–80 (1990). performed biochemical analyses; K.T. performed spectroscopic analyses; A.O.
10. Takahashi, H., Iwai, M., Takahashi, Y. & Minagawa, J. Identification of the mobile purified Fd under the supervision of Y.T.; M.I., K.T. and J.M. designed the study,
light-harvesting complex II polypeptides for state transitions in Chlamydomonas analysed data, and wrote the paper. M.I. and K.T. contributed equally to the study.
reinhardtii. Proc. Natl Acad. Sci. USA 103, 477–482 (2006). The whole study was supervised by J.M. All authors discussed the results and
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and is essential for photoprotection in Arabidopsis. Cell 110, 361–371 (2002). www.nature.com/reprints. The authors declare no competing financial interests.
13. Gulis, G., Narasimhulu, K. V., Fox, L. N. & Redding, K. E. Purification of His6-tagged Correspondence and requests for materials should be addressed to J.M.
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doi:10.1038/nature08885

METHODS the sample (IS) and reference (IR) as –log(IS/IR). The measuring beam was pulse-
Strains. C. reinhardtii wild-type strain 137c and the PSI-His mutant in which a modulated (20 ms duration), and the sampling interval was varied from 0.3 to
His-tag is fused to the N terminus of PsaA13 were grown under 20 mmol photons 100 ms to provide enough time resolution to avoid the actinic effect by itself.
m22 s21 in Tris-acetate-phosphate medium27 aerated with ambient air at 23 uC. Measurements were repeated three to 10 times and averaged.
A mutant deficient in PSI, DPSI, was generated biolistically with a pBD302 Spectroscopic analyses. P700 oxidation in State 1 and State 2 cells was estimated
plasmid containing an inactivated psaA gene. The DPSI and the Cyt bf-deficient from DA810 (Fig. 1c). Cells were exposed to actinic illumination by white LED
mutant FuD6 (ref. 28) cells were grown in the same way as wild-type cells, but (4,400 mmole photons m22 s21) for 200 ms after 30 s dark incubation with 10 mM
kept in the dark. DCMU and additional inhibitors (20 mM AA, 40 mM DBMIB). For measuring
Sucrose density gradient ultracentrifugation. Induction of State 1 with DCMU redox difference spectra (Fig. 2b), the A4 fraction was dissolved in 25 mM MES
and State 2 with FCCP, isolation of thylakoid membranes, and solubilization of (pH 6.5), 0.03% TM and 1 mM 5-methylphenazonium methosulphate (final
the membranes with 1.0% (w/v) TM (Anatrace) were done as described previ- 40 mg ml21 chlorophyll). The oxidised (by 400 mM potassium ferricyanide) spec-
ously11. Separation of photosynthetic protein complexes by SDG ultracentrifu- trum was subtracted from the reduced (by 2 mM Na-ascorbate or 2 mM dithio-
gation was also done as described previously10, except that 0.05% TM was nite) spectra. Cyt bf /PSI stoichiometry was calculated from redox difference
included in the gradients, and a P40ST rotor (Hitachi) was used at 91,500 g absorptions of Cyt f (554 nm) and P700 (700 nm) using the extinction coef-
for 24 h at 4 uC. For the NaCl treatment experiment, the heaviest band containing ficients of 26 (ref. 31) and 64 mM21 (ref. 32), respectively. Three batches of
the A4 supercomplex was fractionated, concentrated by Amicon filtration (100 A4 fraction from independent SDG preparations were used for measurements.
MWKO, Millipore), and re-subjected to SDG ultracentrifugation as described For measuring chemical reduction of Cyt b (Fig. 3b), the A4 supercomplex was
above, except that 250 mM NaCl was included in the gradients. suspended in anaerobic reaction buffer (30 mM Tris (pH 7.5), 50 mM NaCl,
Nickel affinity chromatography. Isolation of the His-tagged PSI protein com- 0.03% TM, 5 mM Na-ascorbate (reducing Cyt f), 5 mM glucose, 160 units glu-
plex from PSI-His cells was carried out using a nickel affinity column as cose oxidase (100 mg ml21 chlorophyll)), in which Cyt b was reduced. To this
described previously11, except for the following. State 2 thylakoid membranes solution, 1 mM NADPH, 1 mM Fd (oxidised) or 3 mM dithionite were added as
were first isolated from the PSI-His strain and subjected to SDG ultracentrifuga- indicated, and absorbance changes (peak at 562 nm) were monitored. Cyt b
tion as described above. The obtained A4 band was diluted with sucrose-free photoreduction was examined by giving actinic illumination (red LED,
buffer, concentrated by Amicon filtration (100 MWKO, Millipore), and subjected 460 mmol photons m22 s21) with and without 1 mM Fd (Fig. 3c). The anaerobic
to nickel affinity chromatography. NaCl was removed from all solutions before reaction buffer described above was used together with 10 mM Pc as an electron
subjecting them to the nickel affinity column. donor for P700. Chlorophyll concentration was 200 mg ml21. Cyt b reduction
Antibodies. Antibodies specific for D1, CP47, CP26, CP29, LhcbM5 (LHCII type was estimated from DA562 minus the baseline drift between DA546 and DA577.
II), PsaA and PsaF were described previously10,11. Antibodies specific for PGR5, Kinetic analysis of P7001 re-reduction by Pc was performed by tracing DA700–
PGRL1 and FNR were gifts from T. Shikanai, D. Leister and T. Hase, respectively. DA730 changes after oxidation of P700 by a single turnover flash (10 ms, 635 nm,
Antibodies specific for AtpB, Cyt b6, Cyt f, Fd and PETC were obtained from shown as an arrow head) (Fig. 3e). The A4 fraction was dissolved in 25 mM MES
Agrisera (Sweden). (pH 6.5), 0.03% TM, 200 mM methyl viologen and 5 mM Na-ascorbate
Kinetic spectrophotometer. Kinetic analyses of electron transfer were per- (100 mg ml21 chlorophyll). Pc concentration was varied from 0.1 to 1 mM. The
formed with a custom-made spectrophotometer29,30 modified for dual-beam ionic strength was adjusted to 0.04 by the addition of NaCl. Flash-induced
absorbance measurement. The measuring beam of infrared (ELD-810, oxidation of Cyt f by P7001 was measured in the presence of different con-
Roithner, for DA810 measurements) or white LED (LXHL-PW03, Lumilends centrations of Pc (Fig. 3f). Measuring systems and reaction media were as
Lighting, for all other DA measurements) with a band-pass filter was divided described above. Cyt f oxidation was estimated from DA554 minus the baseline
by a beam-splitter and passed through sample and reference cuvettes. A series of drift between DA546 and DA562.
band-pass filters were used to measure electron transfer in Cyt bf (546, 554, 562, 31. Metzger, S. U., Cramer, W. A. & Whitmarsh, J. Critical analysis of the extinction
577 nm, bandwidth 2–3 nm) and PSI (700 and 730 nm, bandwidth 10 nm). The coefficient of chloroplast cytochrome f. Biochim. Biophys. Acta 1319, 233–241 (1997).
sample cuvette was exposed to actinic illumination (LED or xenon lamp), and 32. Markwell, J. P., Thornber, J. P. & Skrdla, M. P. Effect of detergents on the reliability
the light-induced absorbance change was calculated from the transmittance of of a chemical assay for P-700. Biochim. Biophys. Acta 591, 391–399 (1980).

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