Evolution of photosynthesis

1

• Evolution of photosynthesis
• Accidental use of pigments where disequillibria
  easily exploited.
• - Anoxygenic photosynthesis may have evolved from
  bacterium using infrared thermotaxis.
• - Organism drifted into shallow water used
  sunlight.
• Later evolution of an oxygen generating complex
  exploiting Mn4O4 to
• Mn4O6 chemistry to generate O2 from H2O.
• About 2.7 billion years ago cyanobacteria
  like-things evolved.
• - Genetic exchange between interdependent green
  purple bacteria.
• - Created an organism which could live freely on
  the planet wherever H2O, CO2 light available.
• - Tremendous increase in the biosphere.

2

• Impact on biodiversity
• Photosynthesis made the atmosphere O2 rich.
• - Entire biosphere now rich'' in redox
  potential.
• Evidence that O2 levels increased 2.2 billion
  years ago.
• - Possibly due to complex eukaryotes.
• Cyanobacteria (for real) evolved about 2.0
  billion years ago.
• - First invertebrates about 0.7 billion years
  ago.
• - First plants on land about 0.5 billion years
  ago.
• - First reptiles 0.4 billion years ago.

3

• Classes of reaction centres
• Photosynthetic bacteria, algea plants fix CO2.
• - Produce 10 billion tons of carbohydrate
  annually.
• - Eight times human energy consumption.
• Two types of reaction centre
• - Type-I (green sulphur bacteria) use
  iron-sulphur centres as terminal electron
  acceptor.
• - Type-II use (purple photosynthetic bacteria)
  use quinones as terminal electron acceptor.
• In cyanobacteria, algea plants a more complex
  system.
• - Splits water to produce oxygen transports
  electrons to NAD.
• - Like both type-I \ type-II reaction centres
  acting in series.
• Structurally evolutionary related to bacterial
  reaction centres.

4

• Leaves
• Photosynthesis in plants occurs in leaves.
• - Water carbon-dioxide enter the cells of the
  leaf.
• - Sugar oxygen leave the cells of the leaf.
• Cuticle is a protective waxy-layer.
• - Water transported to the leaf through the
  xylem.

- Stomata (plural for stoma) provide a pathway
for carbon-dioxide to be taken up oxygen to be
released. - Mesophyll cells fill the region
between the epidermis layers contain the
chloroplasts.
5

• Chloroplasts Thylakoids
• Chloroplasts are organelles specific to plants.
• - Approximately 4-10 mm diameter.
• Contain
• - Outer membrane.
• - Inner membrane.
• - The stroma (the matrix within the inner
  membrane).
• - Flattened vesicles called Thylakoids.
• Where energy transduction takes place.
• - Inner thylakoid space usually called the lumen.

6

• Thylakoid membrane
• Thylakoid membrane has two distinct regions.
• - Stacked regions called grana which contain
  photosystem II.
• - Non-stacked regions, called stroma lamellae,
  which contain photosystem I ATPsynthase.

7

• Photosynthesis in green plants
• Electron transport is non-cyclic.
• H2O is oxidised to O2 NADP is reduced to
  NADPH.
• l gt 690 nm ineffective at producing O2.
• - O2 production by a reaction centre absorbing at
  l 680 nm.
• Illumination with l 650 nm l 700 nm
  enhanced the oxygen production over illumination
  with l 650 nm alone.
• - A second reaction centre must absorb at l 700
  nm.
• Two light reactions act in series to encompass
  the O2/2H2O to NADP/NADPH redox span of 1,140 mV.

8

• Electron flow for photosynthetic purple bacteria
• Electrons donated to the membrane quinone/quinol
  pool.
• Quinol donates electrons to the bc1-complex
  (like complex IV of the mitochondria).
• The bc1-complex pumps protons passes the
  electrons to cytochrome c2.
• Cytochrome c2 returns the electrons to the
  reaction centre eventually back to the special
  pair.
• Proton gradient harvested by ATPsynthase to
  generate ATP.

9

• Electron Flow in Plants
• Photosystem II
• Requires l lt 680 nm.
• Abstracts electrons from water raises them to
  sufficiently negative potential so as to reduce
  plastoquinone (PQ).
• bf-complex.
• Accepts electrons from PQ passes them on to
  plastocyanin (like cytochrome c of the
  mitochondria).
• Pumps protons.
• Photosystem I
• Accepts electrons from plastocyanin.
• Reduces ferredoxin, an Fe/S protein.
• NADP is reduced to NADPH from ferredoxin by
  ferredoxin-NADP oxidoreductase.

10

• Structure of PSII (from a cyanobacterium)
• The primary electron donor is P680
• - Formed by two chlorophylls 10 Å apart (c.w.
  bacterial reaction centres, 7.6 Å apart not
  really a special pair).
• Contains two further chlorophylls, pheophytin
  bound plastoquinone sites (as in the bacterial
  reaction centre).
• - Quinone is the electron acceptor (type-II
  reaction centre).
• Contains a 4Mn complex which abstracts electrons
  from water.
• Contains 17 subunits (36 transmembrane helices).
  
• Subunit PsbO stabilises the Mn ions.

11

• Electron flow in PSII
• Electron from photo-excited P680 flows to QA (
  then to QB) via a chlorophyll and a pheophytin.
• - Exactly as with photosynthetic purple bacteria.
• Second electron transfer releases a quinol from
  QB.
• After each charge separation step P680
  abstracts one electron from a nearby manganese
  cluster via a tyrosine residue (Tyrz).
• Four positive charges accumulate on the Mn
  cluster which oxidise two water molecules
  release O2 4H.

12

• Water splitting reaction
• The photo-excited P680 is reduced by a tyrosine
  residue, Tyrz.
• Tyrz in turn abstracts an electron from the Mn
  cluster.
• - Located 7 Å from the Mn cluster.
• Four photon absorption steps lead to 4Mn being
  oxidised to 4Mn.
• Highly electropositive.
• Spontaneously accepts 4 electrons from H2O (Em,7
  of the O2/2H2O couple is 810 mV).
• Most electropositive reaction in nature.
• Centre-to-centre distance from the Mn cluster to
  the P680 chlorophylls is 18.5 Å to PD1 25.1 Å
  to PD2.

13

• Active site of PSII
• Five metal ions in the active site.
• 4 manganese ions 1 calcium ion.
• 3 Mg the Ca at four corners of a distorted
  cube.
• Oxygen atoms at the other corners.
• - The fourth manganese ion is liganded by one
  oxygens of the cube.
• The 31 Mn tetramer predicted from
  spectroscopy studies.
• The three manganese ions in the cubane structure
  have four or five ligands.
• - Indicates that water molecules are bound to the
  cluster.

14
Bicarbonate ion

• Calcium ions ligands are the three oxygens of
  the cube a bicarbonate that bridges to the
  fourth manganese ion.
• Suggested that the bridging bicarbonate may be
  occupying the active site.
• Two substrate water molecules may replace it
  later in the enzyme cycle.

15

• Tyrz
• Believed a tyrosyl radical, TyrZ, acts as a
  hydrogen-atom abstractor (removing both protons
  and electrons) from substrate water.
• TyrZ is only 5.1 Å away, suggesting direct
  oxidation of calcium-bound water by the tyrosine
  radical.
• But no pathway for a proton ejected from the
  tyrosine upon formation of the radical to be
  translocated away.
• Possible that structural readjustment during
  turnover provide a proton exit pathway from the
  TyrZHis190 pair.
• Possible that the proton leaves via another path
  (shown) TyrZ abstracts only electrons from
  water.

16

• Water splitting reaction
• The enzyme accumulates four positive
  charge-equivalents
• Deprotonation occurs to compensate the charge
  accumulation on some steps, before oxidizing 2H2O
  and releasing O2.
• The valence of the Mn ions increases on the S0
  to S1 to S2 steps
• Less certain for the S3 S4 steps.

17

• Electron transfer the bf complex
• Electrons passed from PSII to quinone/quinol
  pool.
• Electrons from the quinone/quinol pool to the
  b6f complex.
• Protons were taken up from the stroma by PSII.
• The bf-complex releases protons to the lumen.
• Pumps additional protons using the Q-cycle.
• bf-complex delivers electrons to plastocyanin.
• Platsocyanin delivers electrons to photosystem I.

18

• Structure of PSI (from a cyanobacterium)
• The primary electron donor is P700.
• - Formed by two chlorophylls 6.3 Å apart.
• Two major subunits similar to those of PSII.
• Contain two further chlorophylls two
  pheophytin.
• All photosystems must have the same evolutionary
  origin.
• Bound plastocyanin is the electron donor.
• An iron-sulphur complex Fe4S4 (one of three) is
  the terminal electron acceptor (type-I reaction
  centre).
• Contains 12 subunits (35 transmembrane helices).

19

• Electron flow in PSI
• Plastocyanin binds \ donates electrons to
  P700.
• - Em,7 couple for plastocyanin ox/red is 370 mV.
• Electrons flow as bacterial reaction centres,
  but to an an FeS complex.
• Related to green sulphur bacterial reaction
  centres.
• PSI does not pump protons.
• Fe4S4 complex reduces the iron-sulphur protein
  ferredoxin.
• Em,7 of ferredoxin ox/red is -530 mV.
• 1.31 V more electronegative than the original
  donor (O2/2H2O has an Em,7 of 820 mV)
• Reduced ferredoxin in turn reduces NADP to
  NADPH (Em,7 -320 mV) through NADP reductase.

20

• Cyclic electron transfer
• The main destination of the NADP produced by
  non-cyclic electron
• flow is the Calvin cycle.
• Fixes CO2.
• Requires three ATP for every two NADP consumed.
• This (or other factors) can generate an ATP
  shortfall.
• Cyclic electron transfer occurs when electrons
  from ferredoxin are used to reduce plastoquinone.
• Plastoquinone donates electrons to the
  bf-complex.
• The bf-complex pumps protons using the Q-cycle.
• Probably involves a ferredoxin/PQ
  oxidoreductase.
• Cyclic electron transfer provides a mechanism
  for pumping protons ( hence generating ATP via
  ATPsynthase) but does not produce O2 or NADP.

21

• Antenna LHCII
• Reaction centres of both PSI PSII are
  surrounded by antennae
• Two antenna subunits of PSII have 12 14
  chlorophylls respectively.
• The antenna complex of PSI contain 90
  chlorophylls.
• In addition there is a light harvesting complex
  normally associated with PSII (called LHCII).

PSII crystallised as a dimer.
PSI crystallised as a trimer.
22

• Arrangement of the thykaloid membrane
• Folded regions called grana enhanced with PSII.
• - LHCII and bf-complex also present.
• Stroma lamellae enhanced with PSI.
• - bf-complex ATPsynthase also present.

23

• Modification of the thykaloid membrane
• Light quality spectral distribution changes.
• PSII (lmax 680 nm) reduces quinone to quinol.
• PSI (lmax 700 nm) (indirectly, via b6f
  complex) reduces quinol to quinone.
• Changes in the spectrum of the light (more or
  less 680 relative to 700) will change the quinol
  consumption/production ratio.
• Ideally rate of quinol production rate of
  quinol consumption.
• Changes in the ratio of quinol to quinone
  indicates a deviation from optimal kinetics.
• Feedback mechanism sensitive to the
  quinol/quinone ratio.

24

• Phosphorylation of LHCII
• Excess plastoquinol causes LHCII kinase to
  phosphorylate LHCII.
• - Phosphorylation induces a change in
  conformation LHCII migrates out of the grana
  complexes with PSI.
• Excess plastoquinone causes phospho-LHCII
  phosphatase to dephosphorylate LHCII.
• - LHCII migrates into of the grana complexes
  with PSII.
• In complex with PSII, LHCII increases the
  production of plastoquinol
• In complex with PSI the rate of plastoquinol
  consumption is increased.
• - This feedback mechanism maintains the optimal
  balance.

25

• Carbon fixation reactions
• Ribulose-1,5-bisphosphate carboxylase (rubisco)
  catalyzes the addition of gaseous carbon dioxide
  to ribulose-1,5-bisphosphate.
• C5O3H8(PO4 2-)2 CO2 ? 2 C3O3H4PO42-

-
H2O
26

• Most of the phosphoglycerate (C3O3H4PO42-)
  produced is recycled to build more ribulose
  bisphosphate (C5O3H8(PO42-)2).

27

• Calvin cycle
• The production of sugars must be energy
  demanding.
• Otherwise could not be an energy source for the
  cell!
• Fixation of CO2 to ribulose 1,5 biphosphate is
  energetically favourable (but slow) since the
  reactant is highly reactive.
• The Calvin cycle makes 1 molecule of
  glyceraldehyde 3-phosphate and recovers 5
  molecules of ribulose 1,5 biphosphate from 6
  molecules of 3-phosphoglycerate produced by 3
  turnovers of Rubisco.
• Costs 3 ATP and 2 NADPH for each CO2 fixed.
• Equates to 9 ATP and 6 NADPH for each
  glyceraldehyde 3-phosphate produced.
• This is a suger precurser, feeds the plant or is
  stored as starch.

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29

• Rubisco
• The only enzyme capable of fixing CO2.
• Attaches CO2 to ribulose bisphosphate.
• Clips the lengthened chain into two identical
  phosphoglycerate pieces.
• The reaction is slow, turning over 3 CO2 per
  second.
• Rubisco is extremely abundant, 15 of all
  chloroplast proteins.

H2O
30

• Can also consume O2 in a wasteful reaction.
• - Thought to be a relic of when O2 was a minor
  component of the atmosphere very toxic to the
  cell.
• Contains eight copies of a large a small
  protein.
• - Total mass of 550 kDa.
• Is regulated by light.
• In the day-time a CO2 molecule is covalently
  bound to an active site Lysine.
• Enables the active site to form correctly.

31

• Active site of Rubisco
• Arranged around a magnesium ion (green).
• - The magnesium ion is fixed by three amino
  acids, including a modified lysine (an extra CO2
  is attached).
• An intermediate-state analogue is also shown.
• - This analogue could be co-crystallised with
  good occupancy.

32

• Balancing the equation
• Light driven reactions
• 12 NADP 18 ADP 18 P 6 H 48 h n
• ? 6O2 12 NADPH 18 ATP 6H2O
• Dark reactions
• 6CO2 18 ATP 12 NADPH 12 H2O
• ? C6H12O6 18 ADP 18Pi 12 NADP 6 H
• Sums to give overall
• 6 H2O 6 CO2 48 h n ? C6H12 O6 6 O2

33

• Summary
• All photosynthetic reaction centres have a
  common ancestral origin.
• Type I reaction centres deliver electrons to an
  Fe4S4 complex.
• Type II reaction centres deliver electrons to a
  bound quinone.
• In bacteria have cyclic electron flow generate
  ATP.
• In plants cyanobacteria electrons flow from an
  electron donor (H2O) to an eventual acceptor
  (NADP).
• - Pumped protons used to generate ATP via
  ATPsynthase.
• Downstream dark reactions use the NADPH ATP to
  fix CO2.
• - Photosynthesis is the mechanism whereby CO2 is
  fixed and O2 is generated by a series of light
  driven reactions.

34

• Unanswered questions
• What are the structures of PSI PSII from
  plants?
• - How do they differ from their cyanobacteria
  counterparts?
• Why is the charge separation reaction not
  reversible?
• - Must hinge on light-driven structural changes.
• What causes LHCII to migrate in the thykaloid
  membrane?
• - A conformational change?
• What role does the protein itself play in
  electron transport?
• - Is it merely a scaffold or how does it regulate
  electron flow?
• ect etc etc.........