understanding photosynthesis the most important process on the planet John Gray Department of Plant Sciences University of Cambridge

Life on earth depends on plants for photosynthetic CO 2 fixation and O 2 evolution

light 6CO 2 + 6H 2 O  C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O  C 6 H 12 O 6 + 6O 2 energy respiration Photosynthesis a highly efficient energy transduction process conversion of light energy into chemical energy

Cross-section of a leaf 100  m Mesophyll cells

Thylakoid membrane chlorophyll light-harvesting electron transfer O 2 evolution energy production Stroma Rubisco CO 2 fixation sugar and starch synthesis Pea chloroplast 1  m

Schematic chloroplast sealed thylakoid membrane membrane-enclosed stroma

Photosynthetic processes in the thylakoid membrane The Light Reactions

Structures of thylakoid membrane complexes

Light absorption by chlorophylls All chlorophyll is associated with proteins to form light-harvesting complexes in the thylakoid membrane There is no free chlorophyll

Structure of LHCII trimer Kühlbrandt et al. (1994) Liu et al. (2004)

LHCII trimers in grana stack

Light is absorbed by individual chlorophylls in the light-harvesting complexes Energy is transferred from one pigment to another via Resonance Energy Transfer This transfer funnels the energy to a reaction centre where electron transfer starts Energy transfer in light-harvesting complexes

Low resolution structures of photosystem II electron microscopy membrane preparations single particles - negative stain arrangement in thylakoid membrane

Photosystem II - at 3.5Å resolution

D1 and D2 polypeptides - the core of PSII 5 transmembrane spans similar to purple bacterial reaction centre D1 is the product of the chloroplast psbA gene

Prosthetic groups of PSII core

OXYGEN EVOLUTION 2H 2 O  O 2 + 4H + + 4e by analogy to sulphur bacteria (van Niel 1930) H 2 S  S + 2H + + 2e 1970 Joliot and Kok - measured O 2 yield from saturating light flashes O 2 evolution every 4th flash - system for accumulating 4 positive charges

Structure of the manganese cluster 'Dangler' model cubane Mn 3 CaO 4 cluster + fourth Mn linked via O

Photosynthetic electron transfer

ATP synthesis coupled to electron transfer

Structure of ATP synthase ,  and  subunits cross section side view

Mechanism of ATP synthesis NOBEL PRIZE 1997: Paul Boyer (UCLA) Rotational catalysis John Walker (Cambridge) X-ray structure showing 3 different conformations for 3  subunit dimers

Rotary catalysis by ATP synthase

Models of H + translocation proton translocation through a subunit drives rotation of c subunit ring and  subunit b subunits (b and b' in CFo) act as stator to prevent rotation of  subunits

Light reactions of photosynthesis Light absorption by chlorophylls in light-harvesting complexes Electron transfer initiated at reaction centres in photosystem II and photosystem I Electron transfer from H 2 O to NADP + generating O 2 and reducing power Coupled H + liberation in thylakoid lumen provides driving force for ATP synthesis

The dark reactions: capturing CO 2 Light reactions generate ATP and NADPH Provide energy for fixing CO 2 Time before present (billion years) CO 2 O2O2 Rubisco appears Atmospheric partial pressure CO 2 fixation had a massive impact on global climate

The numbers are HUGE Atmospheric CO 2 is 0.035% (and rising!) Total CO 2 in atmosphere 700 x 10 9 tonnes Photosynthesis fixes ~100 x 10 9 tonnes per year ~15% of total atmospheric CO 2 moves into photosynthetic organisms each year! The dark reactions: capturing CO 2

Rubisco is made from 8 small and 8 large subunits Active site Rubisco Ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase catalyses CO 2 fixation into C 3 compounds is the most abundant protein on the planet

CH 2 OP C=O H-C-OH CH 2 OP CO 2 CH 2 OP H-C-OH COOH H-C-OH CH 2 OP + H-C-OH CHO CH 2 OP C=O CH 2 OH Rubisco reaction RuBP C 5 sugar 3-PGA 2 x C 3 acid ATP NADPH 2 x C 3 sugars H2OH2O

starch 6 cycles 6C56C5 12C 3 10C 3 6CO 2 2C32C3 C6C6 export from chloroplast C6C6 sucrose 6C56C5 Regeneration via C 4 C 5 C 6 & C 7 sugar phosphates 12C 3 C6C6 6 ATP 6 NADPH 6 ATP

Photosynthesis Light-driven electron transfer from H 2 O to NADP + generating O 2 and reducing power Coupled H+ translocation into thylakoid lumen used to generate ATP CO 2 fixation into sugars using energy from ATP and NADPH Requires chloroplasts with intact thylakoid membranes

Plant cell stained with DAPI (a DNA fluorochrome)

1  m Chloroplast DNA Each chloroplast contains up to 100 copies of chloroplast DNA Leaf mesophyll cells contains ~100 chloroplasts Leaf mesophyll cells contains ~10000 copies of chloroplast DNA

Genes in land plant chloroplast DNA Rubisco LSrbcL 1 Photosystem IIpsb 13 Cytochrome bfpet 5 Photosystem Ipsa 6 ATP synthaseatp 6 NADH dehydrogenasendh 13 Ribosomal RNArrn 4 (x 2) Transfer RNAtrn ~32 Ribosomal proteinsrpl or rps 19 RNA polymeraserpo 4 Translation initiation factorinfA 1 Acetyl CoA carboxylaseaccD 1 ATP-dependent proteaseclpP 1 Unknownycf

Assembly of photosynthesis complexes chloroplast gene product nuclear gene product All complexes contain at least one nuclear-encoded subunit Requires coordination of plastid and nuclear gene expression

nucleus chloroplast Nuclear gene products structural & regulatory proteins Plastid signals Expression of nuclear genes for chloroplast proteins is regulated by plastid signals reporting the functional state of the chloroplasts Coordination of nuclear and chloroplast gene expression

STROMULES (stroma-filled tubules)

STROMULES stroma-filled tubules interconnecting plastids