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					            Biogenesis and Function of
           Photosystem II, a membrane
           bound water-splitting enzyme


                 Himadri B. Pakrasi
                Department of Biology
                Washington University


Burris2, 2004
       Johnna Roose
      Leeann Thornton
      Yasuhiro Kashino
      Hiroshi Ohkawa



     National Science Foundation
   National Institutes of Health
                Department of Energy
Burris2, 2004
                      Global Photosynthesis




                              Carbon Dioxide




                                                     Oxygen
                                light
                6CO2 + 6H2O                    C6H12O6 + 6O2



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                Where is photosynthesis taking place?



                                 Chloroplast thylakoid membranes




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                Photosynthesis in Chloroplasts




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                Cyanobacteria are like free-living chloroplasts

                                              Thylakoid



                                                Cytosol




                     Chloroplast                          Cyanobacterium

                                   • Size, 2-10 m diameter
                                   • Genomic material
                                   • Thylakoid membrane system

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                Synechocystis 6803
                         • Unicellular cyanobacterium
                         • Both photosynthetic and
                           heterotrophic growth
                         • Facile gene replacement and
                           other genetic tools
                         • First photosynthetic
                           organism with a completely
                           sequenced genome (1996)
                         • 3.6 Mbp genome + 7 plasmids
                         • 3754 genes
                            3264 - chromosomal
                             490 - plasmid
                         • 3575 unique proteins


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                Photosynthetic Redox Reactions




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                                         Photosystem II

                     Stroma/Cytoplasm
                                                           Fe2+
                                                 QA                QB
                                                                                      PQ
                                                                                           PQ
                                                     Phe                        PQH2

                                                                               PQH2        PQ
                                                D1        P680     D2
                                                                                      PQ

                                                      Tyr
                                                     Mn           Mn
                     Lumen                       Ca Mn Mn Cl


                                        2 H2O                             O2
                                                      PsbO
                                                                        4 H+
                                                      OEE


                • A Mn-Ca-Cl cluster in the thylakoid lumenal domain is
                  essential for water oxidation.
                • Lumen-localized soluble extrinsic proteins regulate
                  oxygen-evolution activity of PSII.

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                PSII in Thylakoid Membrane


                Stroma/
                          CP43               CP47
                Cytoplasm
                             D2         D1




                                  4Mn   Cl
                Lumen        Ca


                             PsbO




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                One Step Purification of PSII by
                Metal Affinity Chromatography


                                                      His Tag
                  Stroma/
                            CP43               CP47
                  Cytoplasm
                               D2         D1




                                    4Mn   Cl
                  Lumen        Ca


                               PsbO




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                                                       Purity of the PSII
                                                       Preparation
                                               1.2
                                                       Fluorescence Emission at
                                                                       693.0 nm

                                                1
                                                       77K    684.0 nm
                Fluorescence (relative unit)




                                               0.8

                                                                                    694.0 nm
                                               0.6           683.5 nm


                                               0.4


                                               0.2


                                                0
                                                 600        650               700                 750                 800

                                                                        Wavelength (nm)


                                                                                               Purified PS II
                                                                                               Solubilized Membrane


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  Two Methods for Identification of Proteins using
  Mass Spectrometry (Kashino et al. Biochemistry, 2001)
 Matrix-Assisted Laser Desorption Ionization (MALDI)
        PerSeptive Biosystems Voyager DE-STR MALDI-TOF
        – Low-level (sub-picomole) peptide mass mapping
        – Yields molecular weights of proteolytic peptides
        – High throughput analysis (~1 minute per sample)
        – High mass accuracy ( <50ppm)
        Search protein sequence database with peptide masses to identify protein

 Nano-Electrospray Tandem Mass Spectrometry (ES/MS/MS)
        Micromass Q-Tof Electrospray TOF
        – Low-level (sub-picomole) peptide sequencing
        – Yields MS/MS (sequence) information of any peptide
        – Low throughput analysis (20-60 minutes per sample)
        – Can be automated using nano-LC/MS/MS
        Search database with fragment ion masses to identify protein

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                                               Protein Identification

                                 excise             destain           trypsin          extract
                                 protein                              digest           peptides
                                 spot
                                                                                            spot
                                                                                           MALDI
        rel. abundance




                                665          1501                                           plate
                          515                                 hn
                                       814                                  MALDI
                         500                        2000   ~1 min/spot      analysis
                                      m/z                                                 100 spots/plate
                  Mass Spectrum                                                          (up to 400/plate)
                               search database                                         Determine
                               w/ peptide masses                                YES      PTMs
                                                              Protein ID?
                               search database
                               w/ fragment ion data
                                                              NO
      ES/MS/MS
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                                                MALDI MS of Spot 3
          25000




                                                                                                                1604.7975
                   B
                                                                                                   T6                         Matches sll0470
                   825.1388




          20000




                                                                                                                              Tx = Tryptic peptide matched within 20ppm
          15000                                                                                                               B = Background ion
                       841.1136




                                  B
 Counts




                                                                                       B
                                                                                       1475.7970



          10000
                                                                                                                                                            T13
                                                                         T20
                                                             1277.7604




                                                                                                                1603.7791




                                                                                                                                                              2108.0356
                              861.1238




                                                                           1420.8394




                                                                                                   T17                                                                       B
                                                 1179.6529




                                                                                                    1544.7767




                                                                                                                                                                             2239.1593
                                                                                                                                                1994.0746
           5000




                                                                                                                                                                                         2384.0963



                                                                                                                                                                                                     2510.2023
                                                                                                                            Mass (m/z)


                  800                    1000        1200                1400                                   1600                     1800   2000                      2200             2400                  2600



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                                 MS/MS of T17 from Spot 3
           K               D               W             V            V            D             E            L             E              L       N      G       Q
                                                                                                                                                                      1526.64
     100




                                                                                                                                                                          1544.65
                                                    448.18
                                                     y2

                                           368.17                                                                 1003.43
                                                                                                                    y7           1132.46
                                 283.08                                                                                            y8
      %
                                                                                                     890.36
                                                                                                      y6

                                      300.11                 525.18
                            262.12     b3 396.16
                             y1
                                                                  547.25
                                                                   y3
                        186.07                                                          761.32
                                                                          646.30         y5
                         b2
                                                                           y4                     881.34
                 147.10                                                                                                                                                   1545.65
                                                                                                                         1114.43       1245.53
                   y                                                                                                                                  1359.54
                       243.11                                         638.27                                                    1133.50 y9                    1416.58
                                                      524.19                              767.28            980.34                                      y10
                                                                                                                                                               y11
               141.06                                                                                                    1079.43
                                                                               685.33                      963.45                                1265.44                  1549.84
                                                                                            853.31                                                             1492.63
                                                                                                                                       1197.51
       0                                                                                                                                                                           M/z
               100        200        300       400       500          600        700        800       900         1000          1100    1200      1300    1400     1500         1600



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                           Polypeptides in the purified PSII complex
                                                                                     Mr (kDa)

                Polypeptides that are known to be associated w ith PS II

                     1.    CP4 7 (psbB)                                               45
                     2.    CP4 3 (psbC)                                               34
                     3.    Mn -stabiliz ing protein MS P (psbO)                       31
                     4.    D2 ( psbD1, psbD2)                                         29
                     5.    D1 ( psbA2, psbA3)                                         27
                     6.    Cytochrome c550 (psbV)                                     16
                     7.    Psb28 * (sll1398)                                          10
                     8.    PsbU ( psbU)                                               10
                     9.    Psb27 * (slr1645)                                           9.1
                     10.   Cytochrome b559 large subunit (psbE)                        7.8
                     11.   PsbH ( psbH)                                                5.7
                     12.   PsbZ * (ycf9, sll1281)                                      4.9
                     13.   Cytochrome b559 smal l subunit (psbF)                       4.9
                     14.   Psb I (psbI)                                                4.6
                     15.   Psb L (psbL)                                                4.6
                     16.   PsbT c* (smr0001)                                           4.2
                     17.   PsbJ ( psbJ)                                                4.0
                     18.   Ps bM (psbM)                                                3.8
                     19.   Ps bX (psbX)                                                3.8
                     20.   Ps bK (psbK)                                                3.6
                     21.   Ps bY (psbY)                                                3.6

                Other polypeptides w it h known functions

                     22.   FtsH protease (slr0228)                                    59
                     23.   FtsH protease (slr1604)                                    57
                     24.   Lysyl-tRN A synthetase (lysS, slr1550)                     51
                     25.   Citr ate synthase (gltA, sll0401)                          42

                Nove l polypeptides
                                                Sequence Similarity
                     26.   Sll1414              ORF in Arabidopsis                    24
                     27.   Sll1252              ORF in Arabidopsis                    24
                     28.   Sll1390              ORF in Arabidopsis                    21
                     29.   Sll1418              Extr insic Ps bP protein in plants    19
                     30.   Sll1638              Extr insic Psb Q protein in plants    12
                     31.   Sll1130              ORF in Ric e                          10


Burris2, 2004
                       Evolution of PSII: the paradigm is shifting




                                                                   Hankamer et al, 2001 FEBS Lett.
                                              In this Picture
          The oxygen evolving complex (OEC) of PSII in higher plants and green algae has the 33
          kDa PsbO, 23 kDa PsbP and 17 kDa PsbQ subunits. In cyanobacteria, PsbP and PsbQ
          are replaced by the 10 kDa PsbU and 16 kDa PsbV (cytochrome c550) proteins.

                                        Discontinuous Evolution

Burris2, 2004
     Identification of PSII associated proteins                         PSII


          High resolution gel system to separate PSII proteins

          N-terminal sequencing and MS/MS for identification

                • 21 proteins known to be associated
                  with PSII
                • 5 other proteins of known function
                • 4 unidentified proteins
                • 5 novel PSII associated proteins

                 Polypeptide    Sequence Similarity
                 19a. Sll1414   ORF in Arabidopsis
                 19b. Sll1252   ORF in Arabidopsis
                 18. Sll1390    ORF in Arabidopsis
                 12. Sll1638    Plant PSII oxygen evolving
                                complex protein, PsbQ
                 11. Sll1130    ORF in Anabaena



                                     Kashino et al, 2002 Biochemistry
Burris2, 2004
                    Evolution of PSII: the paradigm is shifting




                Maybe there should be more to this picture….



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                    Mature protein sequence similarity for PsbQ
                       from Synechocystis and Arabidopsis

                Signal Peptide             Mature Protein
                                                                  Arabidopsis

                                                                  Synechocystis

                                        Identity: PsbQ 32.9%




                     MSRLRSLLSLILVLVTTVLVS
                     C
                     Signal peptidase II cleavage leaves a
                      lipid anchor on the Cysteine.

          In plants, PsbQ association with PSII is dependent on the presence of PsbP.

          Is PsbP also present in Synechocystis 6803?



Burris2, 2004
                Identification of PsbP in Synechocystis 6803


            • Amino acid sequence from the mature PsbP
            protein (P82538) was used as a query against the
            Synechocystis 6803 predicted ORFs
            • One gene was found, 33% identity, 49%
            similarity
            • Synechocystis 6803 ORF sll1418 is now called
            psbP


      Thornton, Ohkawa, Roose, Kashino, Keren and Pakrasi (2004) Plant Cell 16: 2164-2175

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      Sequence Comparison of PsbP and PsbQ proteins from
                Synechocystis and Arabidopsis




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      Inactivation of psbP and psbQ genes in Synechocystis




Burris2, 2004
            •PsbP can accumulate in the absence of PsbQ
            •PsbQ can accumulate in the absence of PsbP
Burris2, 2004
                                         Photosystem II

                     Stroma/Cytoplasm
                                                           Fe2+
                                                 QA                QB
                                                                                      PQ
                                                                                           PQ
                                                     Phe                        PQH2

                                                                               PQH2        PQ
                                                D1        P680     D2
                                                                                      PQ

                                                      Tyr
                                                     Mn           Mn
                     Lumen                       Ca Mn Mn Cl


                                        2 H2O                             O2
                                                      PsbO
                                                                        4 H+
                                                      OEE


                • A Mn-Ca-Cl cluster in the thylakoid lumenal domain is
                  essential for water oxidation.
                • Lumen-localized soluble extrinsic proteins regulate
                  oxygen-evolution activity of PSII.

Burris2, 2004
                Both calcium and chloride
                deficiencies retard photoautotrophic
                growth of psbP and psbQ mutant
                strains. In contrast, psbV mutant
                can not grow under such conditions.




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                PSII activity of cells as a function of light intensity


                       BG11                      -CaCl2
                                                                      WT




                                                                      P

                                                                      Q




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          Analysis of Purified PSII preparations




                PsbQ:CP47 = 1   PsbP:CP47 = 0.03

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     •Lower abundance of
     cyt c550 (PsbV) and
     PsbU in isolated PSII
     from the psbQ mutant
     •No change in the
     abundance of PsbO




Burris2, 2004
  Difference absorption spectra of purified PSII complexes


                 0.025




                  0.02




                                                                              Solid line - PSII from psbQ
   Absorbance




                 0.015


                                                                              Dotted line - WT PSII
                  0.01
                                                                              Dashed line - CaCl2 washed WT PSII

                 0.005




                    0
                                                                            Suggest physical interactions
                     540   545   550    555   560   565   570   575   580
                                                                            between PsbQ and cyt c550
                                                                            (PsbV)
                                       Wavelength (nm)




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                                    His Tag

                CP43               CP47

                       D1     D2




                       PsbO
                               V   Q
                              U




Burris2, 2004
                             2.0
                                        Growth of double mutants
                                       WT6803
                             1.8
                                       WT6803+Glucose
                             1.6
                                       QV
                             1.4
                                       QV+Glucose
                O.D. 730nm




                             1.2
                                       QU
                             1.0
                                       QU+Glucose
                             0.8

                             0.6

                             0.4

                             0.2

                             0.0
                                   1             2          3         4   5
                                                        Time (Days)


          psbQpsbV mutant has ~20% PSII-mediated O2-evolving activity


Burris2, 2004
  Inhibitory Effects of hydroxylamine on PSII preparations




                                                            WT

                                                           psbQ




                Vander Meulen et al. (2002) Biochemistry 41:958-966
Burris2, 2004
      Many oxygenic photosynthetic organisms contain PsbP and PsbQ
                                                   100 Indian Mustard
            PsbP                             84
                                                  96   White Mustard                        PsbQ                                          C. reinhardtii
                                                          Arabidopsis PsbP1                                      100
                                            71            Arabidopsis PsbP2                      52                                       Volvox

                                                          Tomat o                                                                               Cyanidium caldarium
                                        44        70
                                                          Tobacc o                                                                         Maize PsbQ1
                                                                                                                                    100
                                                          Spin ach
                                       99         73                                                                                        Maize PsbQ2
                                                          Pea
                                                                                            91
                                                           Wheat                                                                              Spinach
                                 98          98                                                                             42
                                                          Rice                                                                             Arabidopsis Ps bQ2
                                                                                                                       78           58
                 100                    100                 Physcomitrella patens                                                          Arabidopsis Ps bQ1
                                                           Physcomitrella patens
      58                                                                                                                                      Onobrychis
                                                           C. reinhardtii                                    100               99
                                                          Gloeobacter                                                                       Pea

                 100                                    Arabidopsis P82538                                                                 Physc omitrella patens
                                                                                                                          68
                                                        Physcomitrella patens                                                                Physc omitrella patens
                                 100                   Anabaena
                                                                                                                                             Anabaena
                           98                          Nostoc                                                      100
           96
                     60                            Trichodesmium                                            75                             Nos toc
                 41                                      Synechocystis                                 82                                  Tric hodesmium
                                                        T. elongatu s                             85                                       Synec hocystis
                97
                                85                        Synechococcus                     98                                                T. elongatu s
                          100                            P. marinus MIT9313
                                                             P. marinus Med4                                                                   Synec hococcus

                                                                    M. thermautotrophicus                                                         C. tetani
                     0.1 changes                                                                            0.1 changes


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      Evolution of PSII : Possible scenarios




Burris2, 2004
   •    Maitrayee
        Bhattacharyya
   •    Ted Drury
   •    Stacy Hood
   •    Nir Keren
   •    Michelle Liberton
   •    Hiroshi Ohkawa
   •    Johnna Roose
   •    Xuemei Si
   •    Leeann Thornton




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Many oxygenic photosynthetic organisms contain PsbP and PsbQ




Burris2, 2004
                             A closer look at the thylakoid membrane



                                       Carbon                        ATP
                                                                                       ADP
                                                             NADPH


                                                                             ATP
                         H+              H+             Fd     FNR
  Stroma                                                                   synthase
                                                                                      Photosystem II (PSII)
                 PSII          PQ                                                     Plastoquinone (PQ)
                        e-
                               PQH2
                                       PQ
                                              Cyt bf           PSI                    Cytochrome b6f (Cyt bf)

                                      PQH2                                            Plastocyanin (PC)
                                                                                      Photosystem (PSI)
                                                                                      Ferredoxin (Fd)
  Lumen                                                       PC
                                                   PC                         H+      Ferredoxin NADPH
                                             H+                                       oxidoreductase (FNR)
          2H2O               O2 + 4H+




Burris2, 2004

				
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