Photosynthesis

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• Photosynthesis
• Step 1 conversion of light into redox energy.
• Photosynthetic organisms convert light to
  chemical energy.
• - Absorption of a photon changes a component's
  affinity for electrons from relatively
  electropositive (an electron acceptor) to highly
  electronegative (an electron donor).
• Step 2 electron flow.
• Through a cyclic pathway (eg. photosynthetic
  bacteria, electrons flow back re-reduce the
  original donor).
• Through a non-cyclic pathway (eg. Thylakoid
  photosynthesis, electrons are transferred
  uphill'' from H2O to NADP).
• Step 3 ATP produced via a proton gradient,
  analogous to mitochondria.
• - Energy transducing membranes contain
  ATP-synthase.

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Redox potentials
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• Structure of a Bacterial Reaction Centre
• Reaction centre from Rhodopseudomonas viridis is
  built up from four sub-units (L, M, H a
  cytochrome).
• - L (258 amino acids) M (273 amino acids) have
  25 sequence identity.
• - Reaction centre contains four
  bacteriochlorophyll molecules (two of which form
  the special pair'') two bacteriopheophytin
  molecules (chlorophyll molecules without Mg2)
  two quinone molecules one Fe atom.
• - The functional role of the Fe is (probably) to
  stabilize the four-helix bundle (removal does not
  affect electron transfer).
• - The cytochrome subunit has four bound hemes
  delivers electrons to the special pair when
  oxidised).
• First membrane protein structure solved
  Deisenhofer et al., Nature 318, 618 (1985).
• Nobel prize in Chemistry in1988.

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• The special pair''
• The bacterial reaction centre of Rhodobacter
  sphaeroides contains
• a chromophore which absorbs at 870 nm.
• - Formed by two chlorophyll molecules
  approximately 7.6 Å apart.
• - Labeled the special pair'', P870.
• A chlorophyll molecule is like a heme but
  contains an Mg2 (rather than Fe2).
• Absorb light in the visible region because they
  have conjugated double-bond systems.

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• Electron flow in the Bacterial Reaction Centres
• Em,7 of the P870 /P870 couple is 500 mV (ie.
  electropositive).
• Photoabsorption leads to an electronically
  excited species P870 which has an Em,7 -550 mV
  (ie. electronegative).
• Within 10 ps one electron moves to a
  bacteriopheophytin molecule (Bpheo or BP).
• - Like chorophyl but with Mg2 replaced by two
  protons.
• - Pathway via a chlorophyll which flanks the
  special pair.
• Within 200 ps the electron is transferred from
  Bphe- to a bound ubquinone (UQ), making a
  semi-quinone.
• - Named the QA site.
• Approximately 20 ms later the electron is
  transferred from the QA UQ, to the UQ at the QB
  site.

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• Second half of electron flow
• The oxidised special pair P870 relaxes to
  its ground state (rather quickly) and becomes
  P870.
• Since the Em,7 of the P870 / P870 couple is
  500 mV (ie. electropositive) it can be reduced by
  a cytochrome c2 (Em,7 of the cytochrome c2 ox/red
  couple of 340 mV).
• Photo absorption leads to an electronically
  excited species P870 and again a second
  electron is transferred to the boundsemi-quinone
  at the UB site as above.
• The bound UQ2- at site B then takes up two
  protons from the bulk phase, and is released at
  UQH2 (ie quinol) into the lipid bilayer.

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• Stabilisation of semi-quinone?
• Structural studies of the light-adapted reaction
  centre show movements of quinol at QB site.
• - Quinone accepts an electron and is reduced.
• - H-bonding changes.
• - Becomes more tightly bound reducing the danger
  of a highly reactive semi-quinol being released
  to membrane.
• Not clear this study is truely physiological
  (ie. not steady-state conditions).

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• What happens to the electrons which reduce
  quinone to quinol?
• Electrons donated to the membrane quinone/quinol
  pool.
• Quinol donates electrons to the bc1 complex
  (complex IV, of the mitochondria).
• The bc1-complex pumps protons passes the
  electrons to a cytochrome c2.
• Cytochrome c2 returns the electrons to the
  reaction centre eventually back to the special
  pair.

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• Proton pumping by the bc1-complex
• Structurally functionally related to the bc1
  complex of the mitochondria respiratory chain.
• Two quinol binding sites in the bc1 complex.
• - The Q0 site near the P-side of the membrane.
• - The Q1 site near the N-side of the membrane.
• One electron from the Q0 quinol binding site to
  the Q1 quinone binding site, and one (via the
  Reiske protein) to cytochrome c.
• - Oxidation of UQH2 to UQ at Q0 releases two H.
• - Reduction of UQ to UQH2 at Q1 takes up two H.
• Overall two protons are taken up from the
  N-side four protons are released to the P-side,
  for every two quinol molecules to bind (Q-cycle).

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• Adding up protons
• For the bacterial reaction centre every two
  photons cause two protons to be taken up from the
  N-side at the QB site.
• Each quinol molecule to bind to the Q0 quinol
  binding site of the bf-complex causes
• - Two protons to be released to the P-side.
• In addition one electron flows to a quinone at
  the Q1 site.
• - For every two UQH2 consumed at the Q0 site one
  UQH2 is recovered at the Q1 site.
• - This can in turn be consumed at the Q0 site.
• - The overall effect is that two further protons
  are released to the P-side taken up from the
  N-side per quinol generated by the bacterial
  reaction centre.

• Totals two protons pumped per photon absorbed.

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• What happens to the proton gradient?
• Proton pumping creates a proton-motive
  potential.
• This potential is harvested by ATP-synthase.

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• Antennae
• The reaction centre can turn-over 100 cycles per
  second.
• - Even in bright sunlight photon-absorption
  rates insufficient.
• Antennae complexes capture photons transfer
  the energy to the reaction centre.
• - Absorbed light over a wide range of wavelengths
  lt 870 nm by an assembly of polypeptides with
  attached pigments.

Chlorophyll
Carotene
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• Light harvesting complex 1 (LH1)
• - Forms the so-called core'' complex,
  intimately associated with the reaction centre.
• - Contains 24 bacteriochlorophyll 24 carotenoid
  molecules.
• Light harvesting complex II (LHII).
• - Arranged surrounding the LH1 core.
• - Nine transmembrane a-helix a-apopproteins are
  packed side-by-side to form a hollow 18 Å
  cylinder.
• - Nine transmembrane b-helix b-apopproteins form
  an outer cylinder of 34 Å radius.
• - Contains 18 bacteriochlorophyll 9 carotenoid
  molecules.

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• Structure of LHCI
• Showed a ring arrangement.
• - How does QB move in out?
• Identified Puff-X protein.
• - Provides a pathway for QB to move in out.
• - The gap in chlorophyll arrangements not
  problematic for energy storage.

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• Energy transfer to the reaction centre
• Over 99 of bacteriochlorophyll molecules
  absorb light at shorter wavelength than the
  reaction centre.
• - First excited state of a bacteriochlorophyll
  lasts 1 ns.
• - Energy is rapidly transferred to the reaction
  centre.
• Resonance energy transfer.
• - Depends on an overlap between fluorescence
  emission spectrum of the donor excitation
  spectrum of an acceptor.
• - Affected by relative orientations of donor
  acceptor.
• - Occurs on about 1 ps time scale over a 2 nm
  distance.

• Delocalised exciton coupling.
• - Occurs over distances lt 1.5 nm.
• - Direct interactions between molecular orbitals.
• -Very rapid energy transfer.

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• Bright light
• Levels of LH2 controlled by molecular mechanisms
  within the cell.
• - Takes several hours.
• Change from cloudy day to sunlight problematic.
• - Supply of electrons from cytochrome c rate
  limiting?
• - Rebinding of quinone to QB rate limiting?
• Futile re-reduction of P870 pumps in lots of
  heat can cause problems.

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• How is an electron stabilised at QA?
• Illuminated crystals at 100 K recorded
  structural differences.
• Recorded X-ray diffraction observed structural
  changes.

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• Observed movements model
• Clear movements in the cytoplasmic domain of
  subunit H.
• Suggest an opening of a water filled-cavity.
• - Change dielectric properties within the
  protein.
• Cluster of four histidine residues implicated.
• - Take up protons lower electrostatic energy of
  QA-.
• Need about 15 kJ/mol stabilisation seems
  reasonable.

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• Summary
• The bacterial reaction centre was the first
  membrane protein structure solved by X-ray
  diffraction.
• Energy transduction by photosynthetic purple
  bacteria provides a model for cyclic electron
  transfer generation of ATP.
• - Structural details as to why reverse
  electron-transfer reactions are hindered remain
  to be solved.
• Light is efficiently harvested by surrounding
  complexes directed to the reaction centre.
• Basic principles extend to photosynthesis in
  plants.