1. Cytochrome b559 and PsbJ of photosystem II modulate orange carotenoid protein-mediated photoprotection in cyanobacteria
First I like to thank the organizers to give me the opturnity to present our recent work on Qc site of PSII..
朱修安 副研究員
中央研究院植微所
2014/10/25

2. Photosynthetic electron transport chain

3. Regulation of photon capture and the protection and repair of photodamage
Nonphotochemical
Quenching
pp4e-fig jpg
Carotenoids,
Ascobate,
SOD, Catalase
And etc.

4. Proton gradient was used to generate ATP
In high light, too many protons accumulated in the lumen will initiate NPQ in PSII.

5. Structural mechanism of Nonphotochemical fluorescence quenching (NPQ)

6. Current model for blue-light (OCP) mediated photoprotection mechanism in cyanobacteria
Central part of this modle is the orange carotenoid protein (OCP) 1) In darkness and under low light condition the OCP is predominantly in the orange form (OCP o ) and cannot interact with PBS. 2) In high light, the OCP can Absorb blue-green light and induces conformational changes and converting to red form. The red form of the OCP will interact with the APC core and dissipate the energy as the heat ..The other protein called FRP will bind to the red form of OCP and converted it back to orange form. Up to now, there is no report for possible feed back regulation onNPQ mechanism of cyanobacteria.
Kirilovsky & Kerfeld (2011) BBA

7. Electron transfer pathway in PSII
Previous studies have proposed that Cyt b559 participates in secondary electron transfers that protect PSII from photoinhibition forming a cyclic electron-transfer pathway within PSII.
In these models, Cyt b559 is thought to donate its electron, via a -carotene molecule (CarD2), to reduce highly oxidized chlorophyll radicals generated in PSII reaction centers under donor-side photoinhibitory conditions. On the other hand, Cyt b559 may accept an electron from the acceptor side of PSII [QB or PQ] to prevent the formation of damaging singlet oxygen species under acceptor-side photoinhibitory conditions.
Guskov et al (2009) Nat. Struct. Mol. Biol.

8. The QC exchange channel in PSII
These are two PQ exchange chanel was identified in the 2.9A crystal structure, one for qb and the other for qc in addition, the head group of QC is located in the region surround with Car, QB, and lipid. The tail of QC is located in a cavity between psbJ and psbe and psbf of cyt b559. The function of QC is still elusive. Could be invoved in QB function or in cyt b559..
In the 2.9Å PSII crystal structure
Guskov et al (2009) Nat. Struct. Mol. Biol.

9. The proposed function of QC site
involve in exchange of PQ on the QB site from the pool.
modulate the redox potential and reactivity of Cyt b559.
Involve in cyclic ET pathway in PSII.
Te fuction of qc is still ot known. People have proposed Qc might invoved in exhange of PQ on the QB site

10. Qc is not detected in the 1.9Å resolution structure.
Studies are needed to clarify the discrepancy about the QC site and determine the exact QC function.
Up to now it is still a unsolved puzzle in PSII.

11. Site-directed mutagenesis on the QC site using Synechocystis 6803G
We construct several site-drected mutageess around the qc site by using synechoystics 6803g. Today we are going to present four of them. We replaced ala-16 with large resiue phe toblock the qc site. For the same reason we replaced th Val 32 of psbF to phe. In addition, there are two polar residues near the QC binding site. We hange these two ser residues to Ala.
Phe
Ala16 PsbJ
Phe
Val32 PsbF
Ser23 PsbE
Ser28 PsbF
Ala
Ala

12. Estimated PSII content
Table 1. General properties of QC mutant cells of Synechocystis PCC 6803
Strain
PS Growth
O2 evolution rate
(% of wild type)
Estimated PSII content WT +
100% ± 6
100% ± 10
A16FJ
97% ± 5
85% ± 8
V32Fβ
95% ± 2
105% ± 6
S23Aα
83% ± 1
104% ± 2
S28Aβ
79% ± 5
100% ± 1
These sde show properties of QC site mutans. All these QC mutant grew photoauttrophically and have high oxygen evolution activity like WT.

13. Time-dependent flash-induced fluorescence yield in the presence and absence of red actinic light
Intensity ~90 E
These mutant cells showed very dintint pattern onf steady fluoescence kinetic during the photo-induction. We think that we are on somthing impotant. But we do no know how to interpret it. Untl one day, I ask my students, why not try blue light illumination.

14. Blue-light induced NPQ
Medium blue actinic light
Strong blue actinic light
Light intensity
~65 µE
~ 250 µE
~ 400 µE
Wild-type
WT mutant showed strong blue light induced NPQ in 250 micro E high blue light and even stronger in 400 micro E blue light. However, Qc mutant show small effct of 250 and 400 micro E blue light innlumntio.
A16FJ
V32Fβ

15. Blue-light induced NPQ
Strong blue actinic light
Medium blue actinic light
~ 250 µE
Light intensity
~65 µE
~ 400 µE
Wild-type
WT cells shows strong NPQ, however, under same condition, all mutant cells only showed very small effect.
S23Aα
S28Aβ

16. Current model for blue-light (OCP) mediated photoprotection mechanism in cyanobacteria
Central part of this modle is the orange carotenoid protein (OCP) 1) In darkness and under low light condition the OCP is predominantly in the orange form (OCP o ) and cannot interact with PBS. 2) In high light, the OCP can Absorb blue-green light and induces conformational changes and converting to red form. The red form of the OCP will interact with the APC core and dissipate the energy as the heat ..The other protein called FRP will bind to the red form of OCP and converted it back to orange form. Up to now, there is no report for possible feed back regulation onNPQ mechanism of cyanobacteria.
Kirilovsky & Kerfeld (2011) BBA

17. Immuno-detection of the OCP proteins in thylakoid membranes
A previous study demonstrated the strong interaction of OCP with thylakoids by detecting OCP-GFP proteins in phycobilisome-associated (MP) and phycobilisome-free (M) membrane preparations (Wilson et al., 2006).
Most of the OCP-GFP proteins were found co-precipitated with the membrane fractions (Wilson et al., 2006).

18. Immuno-detection of the OCP proteins in WT and mutant thylakoid membranes
phycobilisome-free (M)
phycobilisome-associated (MP)

19. A working model for feed-back regulation of QC site on OCP-mediate NPQ in cyanobacteria
Qc site may be a molecular switch
Closed conformation
Qc site is unoccupied
To explain our mutant results, we proposed that qc site may involve in feed back regulation of blue light induced NPQ. Qc may serve as a molecular switch. When Qc site is unoccupied, APC and PSII may be in a close conformation, so red for of the OCP cannot interact with APC core. When Qc site is occupied by reduced plastoquinol, will induced a conformation change on PSII and APC core. APC an PSII will be in a open conformation, which allow red form of OCP can interact with APC core and trigger NPQ.
Qc site is occupied by PQH2
Open conformation

20. OCP-mediate NPQ is not directly regulated by the redox state of PQ pool
Strong Blue light
Strong Blue light
Strong Blue light
+ DBMIB
+ Red light

21. A model for the interaction of APC with PSII
a previous modeling study suggested that stromal site of Cyt b559 which in pink colors may directly interact with the APC core. Perhap the qc site will induced conformational change of cyt b559, in turn to affect conformation of apc with psii.
Barber, et al. (2003)
Qc site mutations
Cyt b559
and PsbJ
PSII and APC

22. Photosynthetic growth at normal conditions

23. Biomass concentration (mg/ml)
Table 2. Photosynthetic growth rates and biomass production in wild-types and mutant cells.
Light intensity
(µE m-2 s-1)
Strain
Doubling time
(hour)
Biomass concentration (mg/ml) 30
Wild-type
17.7 ± 0.1
0.213 ± 0.011
A16FJ
16.3 ± 0.1
0.275 ± 0.017
V32Fβ
16.0 ± 0.1
0.303 ± 0.017 80
9.1 ± 0.1
0.265 ± 0.007
0.325 ± 0.018
8.7 ± 0.2
0.356 ± 0.017
1.31
1.42
1.22
1.34

24. Site directed mutagenesis on the Qc site by using Synechocystis 6803
On the channel entrance
Ser23
To determine the function of QC, we have constructed several site-directed mutagemesis on Ser23 which located on the entrance or exit of QC exchange pocket and the other three mutation are on the QC binding site.
Ser28
Val32
On the Qc site
Ala16

25. The proposed function of QC site
involve in exchange of PQ on the QB site from the pool.
modulate the redox potential and reactivity of Cyt b559.
Involve in cyclic ET pathway in PSII.
Te fuction of qc is still ot known. People have proposed Qc might invoved in exhange of PQ on the QB site

26. Chlorophyll a fluorescence measurement
Fast kinetics (ET of QA to QB or QB-)
. The decay kineti of chlorophyll a fluorescence correspomf to the electron transfer from QA to QB. As you can see, excep one mutant, ser23A alpha mutant, the other 3 mutants showed fluorecence decay kinetics very similar WT.

27. Thermoluminescence measurement
B1 band (S3QB-)
B2 band (S2QB-)

28. TL measurement WT
A16FJ
S28A
V32F
B1 band
(S3QB-)
32±2 ºC
31±1 ºC
28±2 ºC
B2 Band
(S2QB-)
43±1 ºC
39±1 ºC
39±2 ºC
37±1 ºC
The lower peak position of both B-bands indicates a modification in the energetic stability of the electron on QB- and thus a shift of the redox potential of (QB/QB-) redox couple to more negative values.

29. The proposed function of QC site
involve in exchange of PQ on the QB site from the pool.
modulate the redox potential and reactivity of Cyt b559.
Involve in cyclic ET pathway in PSII.
Te fuction of qc is still ot known. People have proposed Qc might invoved in exhange of PQ on the QB site

30. Conclusions
QC site mutant cells showed severe inhibition on blue-light induced NPQ.
modulating OCP-mediated photoprotection in cyanobacteria may increase photosynthetic growth and biomass production for bioenergy application.
Further studies are in the progress to reveal the exact function of the QC site in PSII.
Now come to the conclusion,

31. Acknowledgement 科技部和中央研究院經費支持 Collaborators Lab members 東華大學 黃俊楊 柯學初教授
Dr. Mercedes Roncel
Universidad de Sevilla y CSIC, Spain.
Lab members
黃俊楊
邱宜芳博士
王幸婷
In the end I like to thank people did these work, funding agency and thank you very much for your attenation.
科技部和中央研究院經費支持

32. Electron transfer pathway in PSII
Previous studies have proposed that Cyt b559 participates in secondary electron transfers that protect PSII from photoinhibition forming a cyclic electron-transfer pathway within PSII.
In these models, Cyt b559 is thought to donate its electron, via a -carotene molecule (CarD2), to reduce highly oxidized chlorophyll radicals generated in PSII reaction centers under donor-side photoinhibitory conditions. On the other hand, Cyt b559 may accept an electron from the acceptor side of PSII [QB or PQ] to prevent the formation of damaging singlet oxygen species under acceptor-side photoinhibitory conditions.
Guskov et al (2009) Nat. Struct. Mol. Biol.

33. Chlorophyll a fluorescence measurement
Fast kinetics (QA to QB or QB-)
Charge recombination (+DCMU)
Left hand side show fask kinetic experiments. The decay kineti of chlorophyll a fluorescence correspomf to the electron transfer from QA to QB. As you can see, excep one mutant, ser23A alpha mutant, most mutants showed fluorecence decay kinetics very similar WT. Right hand side shows charge recombinatio experiments. All mutant show decay knnetic very similar WT cells.