University of Groningen

Structure of photosystem IIvan Bezouwen, Laura

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Introduction    Green   organisms   use   a   photosynthetic   apparatus   to   convert   light   energy   into  chemical   energy.   This   photosynthetic   process   involves   four   major   proteins:  photosystem  I  (PSI),  photosystem  II  (PSII),  cytochrome  b6f  and  ATP  synthase.  PSI  and  PSII   are   light   driven   and   perform   the   primary   reactions   of   photosynthesis.   In   this  reaction   water   is   converted   into   oxygen   and   additional   smaller   enzymes   convert  NADP+  to  NADPH.  In  the  dark  reactions  carbon  dioxide  fixation  is  used  for  conversion  of  energy  stored  in  ATP  and  NADPH  into  sugars.  When  PSII  is  excited  by  sunlight,  it  is  able   to   split   water   into   oxygen,   which   is   released   into   the   air,   and   protons   and  electrons.  These  four  proteins  are  embedded  into  the  thylakoid  membranes,  and  in  plants  these  membranes  are  located  a  specialized  organelle,  the  chloroplast.    Both  PSI  and  PSII  are  large  multi-­‐subunit  protein-­‐pigment  complexes.  Depending  on  the  organism  PSI  and  PSII  have  a  membrane-­‐embedded  peripheral  antenna  complex,  the   light   harvesting   complex   (LHCI   or   LHCII),   or   a   membrane-­‐bound   complex,   the  phycobilisome.   The   structure   of   PSI   is   solved   by   X-­‐ray   crystallography   for   several  organisms,   but   for   PSII   only   structures   have   been   solved   for   cyanobacteria   and  recently  for  red  algae,  and  none  for  higher  plants.  Understanding  the  structure  and  therefore   knowing   the   exact   function   is   of   key   importance   for   optimal   use   of   its  properties.    PSI  has  a  core  with  12-­‐16  subunits,  depending  on  the  organisms.  Cyanobacteria  have  the  PBSs  as  an  antenna  complex,  where  in  plants  4  LHCI  proteins  are  present  in  a  1:1  ratio  with  the  core.  Red  algae  are  believed  to  be  the  evolutionary  link,  and  for  PSI  it  is  unknown  how  many  LHCI  are  bound.  PSII  is  a  protein  complex  with  a  dimeric  core.  Depending  on   the  organism   it  has  20-­‐23   subunits.  Whereas   cyanobacteria  and   red  algae  have  the  phycobilisomes  as  an  antenna  complex,  higher  plants  have  the  LHCII  antenna  proteins,  comprised  of  six  different  proteins.  The  three  major  proteins  are  present   as   a   heterotrimer   and   the   other   three   the   minor   proteins,   are   singular  proteins.   The   dimeric   core   can   bind   up   to   six   LHCII   trimers,   from   which   two   are  strongly   bound,   two  moderately   strongly   bound.   Another   two   trimers   are   loosely  bound  and  have  been  up  to  now  only  detected  in  spinach.      In  this  thesis  I  focused  on  a  structural  characterization  of  PSII  of  red  algae  and  higher  plants   using   several   electron   microscopic   techniques.   Ernst   Ruska   developed   the  electron  microscope  in  1931,  and  was  honoured  with  the  Nobel  price  for  physics  in  1986.   The   advantage   of   using   electrons   over   light   is   based   on   their   different  wavelengths.   Electrons   have   a   smaller   wavelength   than   photons   and   therefore  resolutions   in  the  sub-­‐Ångström  range  can  be  reached.   In  the  area  of  biomolecules  

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near-­‐atomic   resolution   structures   are   nowadays   within   reach   due   to   recent  instrumental  developments.    An  electron  microscope  generates  images  from  accelerated  electrons  emitted  by  an  electron   source,   either   a   simple   thermionic   source   or   a   more   sophisticated   field-­‐emission   gun.   A   coherent   beam  of   accelerated   electrons   is   scattered   by   an   object  and  runs  through  a  column  packed  with  a  complex  lens  system  to  produce  a  highly  magnified   image.  To  prevent  unwanted  scattering  events   the  column   is  under  high  vacuum.   To   magnify   the   specimen   several   types   of   lenses   are   necessary.   The  condenser   lens   is   located  before  the  specimen  and   is  used  to  set   the   intensity  and  size   of   the   electron   beam.   The   objective   lens   produces   the   first   image   and   the  projector  lenses  further  magnify  this  image.      For  high  resolution  imaging  several  other  aspects  need  to  be  taken  into  account.  The  first   concerns   imperfections  of   the  microscope   lenses,   causing   several   aberrations:  spherical  and  chromatical  aberrations  and  astigmatism.  The  second  aspect   is  about  the  image  formation  and  how  the  two,  elastic  and  inelastic,  scattering  events  make  the   final   contrast   in   the   image.   The   contrast   transfer   function   (CTF)   is   the   third  aspect.   The   CTF   describes   the   transfer   of   spatial   information   of   the   object   to   the  image,  as  it  is  a  function  of  the  defocus  setting  of  the  objective  lens,  astigmatism  and  the  wavelength  of  the  electrons.    The  final  aspect  to  take   into  account   is  the  camera.  The  final  resolution,  which  can  be  achieved  in  image  processing,  depends  critically  on  the  quality  of  the  camera.  In  the   past   both   film   and   CCD   cameras   were   used,   although   both   had   their  disadvantages.   Slowly   the   direct   electron   detectors   replace   both   the   film   and  CCD  cameras   for  high  resolution  work,  because  of   their   superior   recording  qualities.  An  additional  advantage  is  that  the  images  can  be  recorded  as  dose-­‐fractionated  movies  and  therefore  beam-­‐induced  movements  can  be  corrected.      Even  when  all   these  above  discussed  aspects  are   taken   into  account   the   specimen  still   needs   to   be   prepared   for   the   vacuum.   Two   relevant   techniques   will   be  discussed:   negative   staining   and   ice   embedding.   Negative   staining   occurs   at   room  temperature   and   the   proteins   are   embedded   in   a   heavy   metal   salt   to   avoid  collapsing  and  to  improve  the  contrast.  For  ice  embedding,  also  called  cryo-­‐EM,  the  proteins  are  frozen  fully  hydrated  in  a  thin  layer  of  amorphous  ice,  without  using  a  heavy   metal   salt.   Cooling   to   a   temperature   below   the   sublimation   point   allows  imaging  in  the  microscope  vacuum.    Two   ways   to   solve   structures   with   electron   microscopy   are   discussed,   the   single  particle  reconstruction  and  electron  tomography.  With  single  particle  analysis:  a  2D  or   a   3D   reconstruction   is   made   from   projections   of   single   proteins   or   isolated  

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complexes.  Electron  tomography  is  used  to  study  whole  cells  or  unique  sub-­‐cellular  structures   and   a   3D   reconstruction   of   these   specimens   is   produced.   To   answer  questions   about   the   in   situ   structures   of   complexes   at   higher   resolution   sub-­‐tomogram   averaging   can   be   used.   But   not   all   structural   questions   can   be   solved  using   these   two   techniques   without   introducing   other   specimen   preparation  techniques.  A  helpful   technique  can  be  cryo-­‐focused-­‐ion-­‐beam  milling   for   instance.  With  this  technique  lamella  between  200  and  500  nm  can  be  created,  which  can  be  used   for   electron   tomography.   To   improve   the   signal   to   noise   ratio   for   relatively  thick  specimens,  either  cryo-­‐scanning  transmission  electron  tomography  can  be  used  for  imaging  or  a  phase  plate  can  be  added  for  electron  cryo-­‐tomography.      This  thesis    In  Chapter  2  we  discuss  the  biochemical  and  biophysical  characterization  of  the  red  alga  Cyanidioschyzon  merolae.  Members  of   the   rhodophytan  order  Cyanidiales   are  unique   among   phototrophs   in   their   ability   to   live   at   extremely   low   pH   levels   and  moderately   high   temperatures.   The   photosynthetic   apparatus   of   the   red   alga   C.  merolae  represents  an  intermediate  type  between  cyanobacteria  and  higher  plants,  suggesting  that  this  alga  may  provide  an  evolutionary  link  between  prokaryotic  and  eukaryotic   phototrophs.   Although   we   now   have   a   detailed   structural   model   of  photosystem  II  (PSII)  from  cyanobacteria  at  an  atomic  resolution,  no  corresponding  structure  of  the  eukaryotic  PSII  complex  has  been  published  to  date.  Here  we  report  the  isolation  and  characterization  of  a  highly  active  and  robust  dimeric  PSII  complex  from   C.   merolae.   We   show   that   this   complex   is   highly   stable   across   a   range   of  extreme   light,   temperature,   and   pH   conditions.   By   measuring   fluorescence  quenching  properties  of   the   isolated  C.  merolae   PSII   complex,  we  provide   the   first  direct  evidence  of  pH-­‐dependent  non-­‐photochemical  quenching  in  the  red  algal  PSII  reaction   centre.   This   type   of   quenching,   together   with   high   zeaxanthin   content,  appears   to   underlie   photoprotection  mechanisms   that   are   efficiently   employed   by  this   robust   natural   water-­‐splitting   complex   under   excess   irradiance.   In   order   to  provide  structural  details  of  this  eukaryotic  form  of  PSII,  we  have  employed  electron  microscopy  and  single  particle  analyses  to  obtain  a  17  Å  map  of  the  C.  merolae  PSII  dimer   in   which   we   locate   the   position   of   the   protein   mass   corresponding   to   the  additional   extrinsic   protein   stabilizing   the   oxygen-­‐evolving   complex,   PsbQʹ′.   We  conclude   that   this   lumenal   subunit   is   present   in   the   vicinity   of   the   CP43   protein,  close  to  the  membrane  plane.      In  Chapter   3   an   attempt  was  made   to   solve   a   high   resolution   structure   of   higher  plant  PSII  in  the  membrane  plane.  This  was  done  using  the  barley  (Hordeum  vulgare)  mutant   viridis   zb63.   This   mutant   has   no   PSI   expression,   and   makes   natural   two-­‐

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dimensional  arrays  of  PSII,  with  only  the  C2S2  particle.  These  crystalline  patches  are  suitable  for  structural  analysis.  Initially  a  2D  crystallographic  approach  was  used  for  the  analysis  of   the  PSII   structure.  Due   to  double   layered  patches   in   register,  which  could  not  be   separated,  a   switch  was  made   to  a   single  particle  analysis   technique.  With  both   techniques  a  2D   structure  was   solved  at  a   resolution  of  13  Å.  Based  on  this   result   the   location   and   the   orientation   of   the   minor   antenna   proteins   was  determined.  This  result  was  compared  to  previously  published  results  of  higher  plant  PSII   and   extensively   discussed,   as   well   as   the   use   of   two   techniques   to   solve   this  structure.    Although   the   result   of   chapter   3   was   already   an   improvement   compared   to  previously   published   PSII   maps,   the   structure   was   not   solved   well   enough   to  determine  the  location  of  all  the  subunits.  Therefore  we  decided  to  turn  instead  to  the  largest  most  common  isolated  PSII  supercomplex,  the  C2S2M2  particle.  In  Chapter  4  a  high  resolution  structure  of  plant  PSII  is  presented  for  the  first  time.  Overall  the  structure   is   solved  at  a   resolution  of  5.4  Å,  but   locally   in   the  core   the   resolution   is  close   to   4   Å.   Based   on   this   resolution   it   was   possible   to   locate   almost   all   core  subunits,  including  the  unknown  location  of  subunit  PsbW.  In  the  core  region  there  are   two   intrinsic   small   subunits   missing,   PsbJ   and   PsbY,   as   well   as   the   extrinsic  subunits  PsbP,  PsbQ  and  PsbR.  The  location  and  orientation  of  the  antenna  proteins  was   also  possible,   although   the   resolution  was   limited   for   the  M-­‐trimer   and  CP24.  For  all   regions  we  managed  to   locate  the  chlorophyll  pigments.  The  chlorophylls  of  the   core   region   are   found   to   be   conserved   based   on   the   chlorophylls   of  cyanobacteria.  Based  on  the  crystal  structures  of  the  LHCII  trimer  and  CP29  we  could  also  identify  the  chlorophylls  for  CP26  and  CP24.  Possible  other  pigments  and  lipids  are   indicated   as  well,   but   the  manganese   cluster   is  missing   in   this   structure.   In   an  extensive   discussion   this   is   explained,   and   possible   solutions   to   improve   the  structure  are  discussed.    In   the   last   chapter,  Chapter   5,   the   aim   of   the   study  was   to   determine   the   in   situ  structure   of   the   phycobilisome   and   its   connection   with   PSII   and/or   PSI.  Cyanobacteria  have  no  spatial  separation  of  the  two  photosystems  in  the  thylakoid  membranes.   The   phycobilisomes   are   attached   to   the   membranes.   Different  cyanobacteria  with  a  thickness  that  might  be  suitable  for  electron  cryo-­‐tomography  were   studied.  With   this   technique  we  managed   to   visualize   the   internal   thylakoid  membranes  at  the  tip  of  intact  bacteria.  The  membranes  occur  pairwise  at  a  distance  of  approx.  50  nm.  Between  these  membranes,  we  could   locate  the  phycobilisomes  for  the  first  time  in  intact  cyanobacteria.  A  critical  discussion  is  presented  on  how  to  visualize   internal   structure   in   relatively   large   bacteria   by   electron   tomography,   as  well  as  how  future  instrumental  developments  and  sub-­‐tomogram  averaging  may  be  of  help  to  ultimately  solve  the  interaction  of  PSII  and/or  PSI  with  the  phycobilisomes.