Identifying the roles of protein subunits in solar water-splitting

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Identifying the roles of protein subunits in
solar water-splitting

• Robert Fagerlund, PhD
• Department of Biochemistry
• University of Otago

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Photosynthesis
Solar powered reaction
Water Carbon Dioxide ?(Light) Oxygen
Carbohydrate
Our group is interested in using photosynthesis
to optimize a biomimetic cell a system that
replicates natures efficient model
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Light is captured and energy is transferred to
Photosystem II (PS II), which splits water to
produce Oxygen, Hydrogen and free electrons
Electrons travel through the photosystems to
produce energy rich compounds involved in
creating Carbohydrates from Carbon Dioxide We
are interested in Photosystem II
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Dimeric Structure of Photosystem II from
cyanobacteria
Cytoplasm
Membrane
Lumen
PDB 3BZ1 and 3BZ2
Each monomer has ?19 proteins and ?70
cofactors 4 core proteins, 3 extrinisic proteins
and ?12 peripheral proteins What does this added
complexity give to PS II functionality?
Catalyzes the reaction H2O ? 2H 2e- 1/2O2
Without photosystem II, reaction requires ca
2000 oC
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Photosystem II repair
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Photosynthesis in cyanobacterium Synechocystis
sp. PCC 6803

• Photosynthetic
• Unicellular
• Fast growing, doubling time 12 h
• Genome sequenced in 1996
• Genome easily manipulated

Today I will discuss Photosystem II proteins PsbT
and PsbQ, from Synechocystis
From van de Meene et al., 2005
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Photosystem II interface protein PsbT

• PsbT is a protein at the Photosystem II dimer
  interface
• Investigate the effect of removal

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Photosystem II stability in ?PsbT cells

• Synechocystis cells were grown with radioactive
  amino-acids under medium light intensity
• D1 protein incorporates most radioactivity
  through repair pathway
• Cells treated with bright light
• D1 damage exceeds repair

?PsbT cells had greater D1 turnover, therefore
PsbT appears important for Photosystem II
stability
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Photosystem II activity in ?PsbT cells

• Photosystem II activity was determined by ability
  to produce Oxygen
• Effect of formate and bicarbonate investigated
• Bicarbonate is cofactor of RuBisCO and believed
  to help transfer elections from Photosystem II
  Formate resembles bicarbonate therefore a
  competitive inhibitor

• Formate inhibited oxygen production in ?PsbT
  cells, but recovered with bicarbonate inclusion
• Suggests PsbT is important for efficient electron
  transfer and bicarbonate does have an important
  role in Photosystem II

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Extrinsic protein PsbQ
Cytoplasm
Membrane
Lumen

• PsbQ was not in the Photosystem II structure, yet
  is part of the Photosystem II complex and
  associated with maximum activity
• We decided to determine PsbQ structure using
  X-ray crystallography

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Determining PsbQ structure
PsbQ gene from Synechocystis was cloned into E.
coli PsbQ was expressed in E. coli and
purified Protein was mixed with precipitating
reagents and the protein crystallized -Analogy
of salt crystals in water Good protein crystals
can diffract a X-ray beam -The pattern of which
is used to solve protein structure Computers are
essential! We obtained two PsbQ structures one
with zinc bound and another without
Jackson and Fagerlund et al., 2010 PDB 3LS1
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PsbQ conformation changes
PsbQ rearranges important amino-acids when zinc
is bound Perhaps this is a mechanism important
for PS II function
PDB 3LS0
PDB 3LS1
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Proposal for site of PsbQ interaction
In silico docked PsbQ onto Photosystem II Looks
at possible binding sites based on protein shape
and charge
Cytoplasm
Membrane
Lumen
PsbQ
PsbQ

• PsbQ docked at the extreme ends of Photosystem II
  dimer
• Consistent with other biochemical information
• Next, confirm location!

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Summary
In this talk I showed some characterisation of
Photosystem II that lacked PsbT and presented
structure of extrinsic protein PsbQ Finally Phot
osynthesis is a self-repairing process that uses
solar power to create ENERGY that we can
potentially use Further characterisation of this
process, particularly that by Photosystem II,
could help produce an efficient biomimetic cell
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Acknowledgements
Assoc. Prof. Julian Eaton-Rye PsbT Dr. Fiona
Bentley Roger Young Dr. Hao Luo PsbQ Simon
Jackson Dr. Sigurd Wilbanks
Biochemistry Department, UO New Zealand Marsden
Fund OERC