Course Project Engineering electricity production by living organisms http://biophotovoltaics.wordpress.com/

General principle: Bacteria transfer e- from food to anode via direct contact, nanowires or a mediator. H+ diffuse to cathode to join e- forming H2O

Geobacter species, Shewanella species In Geobacter sulfurreducens Om cytochromes transfer e- to anode.

In Geobacter sulfurreducens pili function as nanowires, but e- are not transferred via cytochromes. Pilin mutants have been isolated.

Nanowires have also been found in Shewanella oneidensis Pseudomonas aeruginosa, Synechocystis PCC6803 in low CO2 (A) and Pelotomaculum thermopropionicum (C)

Plan A: Use plants to feed electrogenic bugs ->exude organics into rhizosphere

Many cyanobacteria reduce their surroundings in the light & make pili

Green algae (Chlorella vulgaris, Dunaliella tertiolecta) or cyanobacteria (Synechocystis sp. PCC6803, Synechococcus sp.WH5701were used for bio-photovoltaics

Energy Environ. Sci., 2011, 4, 4690. Use Z-scheme to move e- from H20 to FeCN, then to anode. H+ diffuse across membrane to cathode, where recombine with e- to form H2O

FeCN is best mediator = electrons come from PSI

conversion of CO2 to ethylene (C2H4) in Synechocystis 6803 transformed with efe gene. Use ethylene to make plastics, diesel, gasoline, jet fuel or ethanol

Changing Cyanobacteria to make a 5 carbon alcohol

Botryococcus braunii partitions C from PS into sugar/fatty acid/terpenoid at ratios of 50 : 10 : 40 cf 85 : 10 : 5 in most plants

Engineering algae to make H2

Engineering algae to make H2

Making H2 in vitro using PSII

Making H2 in vitro using PSI

Making H2 in vitro using PSI & PSII

Using LHCII complexes to make H2 in vitro via platinum Energy Environ. Sci., 2011, 4, 181

PSI and PSII work together in the “Z-scheme” PSII gives excited e- to ETS ending at PSI Each e- drives cyt b6/f Use PMF to make ATP PSII replaces e- from H2O forming O2

Z-scheme energetics

Physical organization of Z-scheme PS II consists of: P680 (a dimer of chl a) ~ 30 other chl a & a few carotenoids > 20 proteins D1 & D2 bind P680 & all e- carriers

Physical organization of Z-scheme PSII also has two groups of closely associated proteins 1) OEC (oxygen evolving complex) on lumen side, near rxn center Ca2+, Cl- & 4 Mn2+ 2) variable numbers of LHCII complexes

PSII Photochemistry 1) LHCII absorbs a photon 2) energy is transferred to P680

PSII Photochemistry 3) P680* reduces pheophytin ( chl a with 2 H+ instead of Mg2+) = primary electron acceptor

PSII Photochemistry 3) P680* reduces pheophytin ( chl a with 2 H+ instead of Mg2+) = primary electron acceptor charge separation traps the electron

PSII Photochemistry 4) pheophytin reduces PQA (plastoquinone bound to D2) moves electron away from P680+ & closer to stroma

PSII Photochemistry 5) PQA reduces PQB (forms PQB- )

PSII Photochemistry 6) P680+ acquires another electron , and steps 1-4 are repeated

PSII Photochemistry 7) PQA reduces PQB - -> forms PQB2-

PSII Photochemistry 8) PQB2- acquires 2 H+ from stroma forms PQH2 (and adds to ∆pH)

PSII Photochemistry 9) PQH2 diffuses within bilayer to cyt b6/f - is replaced within D1 by an oxidized PQ

Photolysis: Making Oxygen 1) P680+ oxidizes tyrZ ( an amino acid of protein D1)

Photolysis: Making Oxygen 2) tyrZ + oxidizes one of the Mn atoms in the OEC Mn cluster is an e- reservoir

Photolysis: Making Oxygen 2) tyrZ + oxidizes one of the Mn atoms in the OEC Mn cluster is an e- reservoir Once 4 Mn are oxidized replace e- by stealing them from 2 H2O

Shown experimentally that need 4 flashes/O2

Shown experimentally that need 4 flashes/O2 Mn cluster cycles S0 -> S4 Reset to S0 by taking 4 e- from 2 H2O

Electron transport from PSII to PSI 1) PQH2 diffuses to cyt b6/f 2) PQH2 reduces cyt b6 and Fe/S, releases H+ in lumen since H+ came from stroma, transports 2 H+ across membrane (Q cycle)

Electron transport from PSII to PSI 3) Fe/S reduces plastocyanin via cyt f cyt b6 reduces PQ to form PQ-

Electron transport from PSII to PSI 4) repeat process, Fe/S reduces plastocyanin via cyt f cyt b6 reduces PQ- to form PQH2

Electron transport from PSII to PSI 4) PC (Cu+) diffuses to PSI, where it reduces an oxidized P700

Electron transport from PSI to Ferredoxin 1) LHCI absorbs a photon 2) P700* reduces A0 3) e- transport to ferredoxin via A1 & 3 Fe/S proteins

Electron transport from Ferredoxin to NADP+ 2 Ferredoxin reduce NADP reductase

Electron transport from Ferredoxin to NADP+ 2 Ferredoxin reduce NADP reductase reduces NADP+

Electron transport from Ferredoxin to NADP+ 2 Ferredoxin reduce NADP reductase reduces NADP+ this also contributes to ∆pH

Overall reaction for the Z-scheme 8 photons + 2 H2O + 10 H+stroma + 2 NADP+ = 12 H+lumen + 2 NADPH + O2

Chemiosmotic ATP synthesis PMF mainly due to ∆pH is used to make ATP -> very little membrane potential, due to transport of other ions thylakoid lumen pH is < 5 cf stroma pH is 8 pH is made by ETS, cyclic photophosphorylation,water splitting & NADPH synth

Chemiosmotic ATP synthesis Structure of ATP synthase CF1 head: exposed to stroma CF0 base: Integral membrane protein

a & b2 subunits form stator that immobilizes a & b F1 subunits a is also an H+ channel c subunits rotate as H+ pass through g & e also rotate c, g & e form a rotor

Binding Change mechanism of ATP synthesis H+ translocation through ATP synthase alters affinity of active site for ATP

Binding Change mechanism of ATP synthesis H+ translocation through ATP synthase alters affinity of active site for ATP ADP + Pi bind to  subunit then spontaneously form ATP

Binding Change mechanism of ATP synthesis H+ translocation through ATP synthase alters affinity of active site for ATP ADP + Pi bind to  subunit then spontaneously form ATP ∆G for ADP + Pi = ATP is ~0 role of H+ translocation is to force enzyme to release ATP!

Binding Change mechanism of ATP synthesis 1) H+ translocation alters affinity of active site for ATP 2) Each active site ratchets through 3 conformations that have different affinities for ATP, ADP & Pi due to interaction with the subunit

Binding Change mechanism of ATP synthesis 1) H+ translocation alters affinity of active site for ATP 2) Each active site ratchets through 3 conformations that have different affinities for ATP, ADP & Pi 3) ATP is synthesized by rotational catalysis g subunit rotates as H+ pass through Fo, forces each active site to sequentially adopt the 3 conformations

Evidence supporting chemiosmosis Racker & Stoeckenius (1974) reconstituted bacteriorhodopsin and ATP synthase in liposomes Bacteriorhodopsin uses light to pump H+ make ATP only in the light

Evidence supporting “rotational catalysis” Sambongi et al experiment a) reconstituted ATPase & attached a subunits to a slide b) attached actin filament to c subunit & watched it spin

Engineering the light reactions?

Engineering the light reactions? Absorb full spectrum

Engineering the light reactions? Absorb full spectrum Improve efficiency of transport w/in PSI & PSII (or recover the energy)

Engineering the light reactions? Absorb full spectrum Improve efficiency of transport w/in PSI & PSII (or recover the energy) Transport electrons to another acceptor From OEE From PQH2 From Fd

Engineering the light reactions? Absorb full spectrum Improve efficiency of transport w/in PSI & PSII (or recover the energy) Transport electrons to another acceptor From OEE From PQH2 From Fd: use hydrogenase to make H2

Engineering the light reactions? Transport electrons to another acceptor From OEE From PQH2 From Fd: use hydrogenase to make H2 or couple Pt catalysts to PSI to generate it directly from ascorbic acid via CytC6

Engineering the light reactions? Transport electrons to another acceptor From OEE From PQH2 From Fd: use hydrogenase to make H2 or couple Pt catalysts to PSI to generate it directly from ascorbic acid via CytC6 Or use N2ase to make H2

under anaerobic conditions, some organisms ferment carbohydrates to facilitate ATP production by photophosphorylation. H2ase essentially acts as a H+/e- release valve by recombining H+ and e- to produce H2 gas that is excreted from the cell

Light-independent (dark) reactions The Calvin cycle

Light-independent (dark) reactions occur in the stroma of the chloroplast (pH 8) Consumes ATP & NADPH from light reactions regenerates ADP, Pi and NADP+

Light-independent (dark) reactions Overall Reaction: 3 CO2 + 3 RuBP + 9 ATP + 6 NADPH = 3 RuBP + 9 ADP + 9 Pi + 6 NADP+ + 1 Glyceraldehyde 3-P

Light-independent (dark) reactions 1) fixing CO2 2) reversing glycolysis 3) regenerating RuBP

fixing CO2 1) RuBP binds CO2

fixing CO2 RuBP binds CO2 2) rapidly splits into two 3-Phosphoglycerate therefore called C3 photosynthesis

fixing CO2 1) CO2 is bound to RuBP 2) rapidly splits into two 3-Phosphoglycerate therefore called C3 photosynthesis detected by immediately killing cells fed 14CO2

fixing CO2 1) CO2 is bound to RuBP 2) rapidly splits into two 3-Phosphoglycerate 3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) the most important & abundant protein on earth

fixing CO2 1) CO2 is bound to RuBP 2) rapidly splits into two 3-Phosphoglycerate 3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) the most important & abundant protein on earth Lousy Km

fixing CO2 1) CO2 is bound to RuBP 2) rapidly splits into two 3-Phosphoglycerate 3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) the most important & abundant protein on earth Lousy Km Rotten Vmax!

Reversing glycolysis converts 3-Phosphoglycerate to G3P consumes 1 ATP & 1 NADPH

Reversing glycolysis G3P has 2 possible fates 1) 1 in 6 becomes (CH2O)n

Reversing glycolysis G3P has 2 possible fates 1) 1 in 6 becomes (CH2O)n 2) 5 in 6 regenerate RuBP

Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell)

Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose

Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose Pi/triosePO4 antiporter only trades DHAP for Pi

Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose Pi/triosePO4 antiporter only trades DHAP for Pi mechanism to regulate PS

Regenerating RuBP G3P has 2 possible fates 5 in 6 regenerate RuBP necessary to keep cycle going

Regenerating RuBP Basic problem: converting a 3C to a 5C compound feed in five 3C sugars, recover three 5C sugars

Regenerating RuBP Basic problem: converting a 3C to a 5C compound must assemble intermediates that can be broken into 5 C sugars after adding 3C subunit

Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C

Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C 4C + 3C = 7C

Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C 4C + 3C = 7C 7C + 3C = 5C + 5C

Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C 4C + 3C = 7C 7C + 3C = 5C + 5C Uses 1 ATP/RuBP

Light-independent (dark) reactions build up pools of intermediates , occasionally remove one very complicated book-keeping

Light-independent (dark) reactions build up pools of intermediates , occasionally remove one very complicated book-keeping Use 12 NADPH and 18 ATP to make one 6C sugar

Regulating the Calvin Cycle Rubisco is main rate-limiting step

Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase : uses ATP to activate rubisco

Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma

Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH: rubisco is most active at pH > 8 (in dark pH is ~7.2)

Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark

Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark Mg2+ moves from thylakoid lumen to stroma to maintain charge neutrality

Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+] c) CO2 is an allosteric activator of rubisco that only binds at high pH and high [Mg2+] also: stomates open in the light

Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma Several other Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also activated by high pH & [Mg2+]

Regulating the Calvin Cycle Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark

Regulating the Calvin Cycle Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark in light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme S - S 2Fdox 2Fdred PSI + PSII light 2e- oxidized thioredoxin reduced SH enzyme (inactive) (active)

Regulating the Calvin Cycle Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark in light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme How light reactions talk to the Calvin cycle S - S 2Fdox 2Fdred PSI + PSII light 2e- oxidized thioredoxin reduced SH enzyme (inactive) (active)

RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate

RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate Releases CO2 without making ATP or NADH

PHOTORESPIRATION Releases CO2 without making ATP or NADH Called photorespiration : undoes photosynthesis

RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon

RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon Both rxns occur at same active site

PHOTORESPIRATION C3 plants can lose 25%-50% of their fixed carbon phosphoglycolate is converted to glycolate : poison!

Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes

Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine produce H2O2

Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria

Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 Why photorespiration loses CO2

Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 5) serine is returned to peroxisome

Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 5) serine is returned to peroxisome 6) peroxisome converts it to glycerate & returns it to chloroplast

Detoxifying Glycolate Why peroxisomes are next to cp and mito in C3 plants Mitochondrion

C4 and CAM photosynthesis Rubisco can use O2 as substrate instead of CO2 [CO2] is 1/600 [O2] _-> usually discriminate well

C4 and CAM photosynthesis Rubisco can use O2 as substrate instead of CO2 [CO2] is 1/600 [O2] Photorespiration increases with temperature

C4 and CAM photosynthesis Rubisco can use O2 as substrate instead of CO2 [CO2] is 1/600 [O2] Photorespiration increases with temperature Solution: increase [CO2] at rubisco

C4 and CAM photosynthesis Solution: increase [CO2] at rubisco C4 & CAM = adaptations that reduce PR & water loss

C4 and CAM photosynthesis Adaptations that reduce PR & water loss Both fix CO2 with a different enzyme

C4 and CAM photosynthesis Adaptations that reduce PR & water loss Both fix CO2 with a different enzyme later release CO2 to be fixed by rubisco use energy to increase [CO2] at rubisco

C4 and CAM photosynthesis Adaptations that reduce PR & water loss Both fix CO2 with a different enzyme later release CO2 to be fixed by rubisco use energy to increase [CO2] at rubisco C4 isolates rubisco spatially (e.g. corn)

C4 and CAM photosynthesis Adaptations that reduce PR & water loss Both fix CO2 with a different enzyme later release CO2 to be fixed by rubisco use energy to increase [CO2] at rubisco C4 isolates rubisco spatially (e.g. corn) CAM isolates rubisco temporally (e.g. cacti)

C4 and CAM photosynthesis C4 isolates rubisco spatially (e.g. corn) CAM isolates rubisco temporally (e.g. cacti) Advantages: 1) increases [CO2] at rubisco

C4 and CAM photosynthesis Advantages: 1) increases [CO2] at rubisco reduces PR prevents CO2 from escaping

C4 and CAM photosynthesis Advantages: 1) increases [CO2] at rubisco reduces PR CO2 compensation point is 20-100 ppm in C3 0-5 ppm in C4 & CAM

C4 and CAM photosynthesis Advantages: 1) increases [CO2] at rubisco reduces PR CO2 compensation point is 20-100 ppm in C3 0-5 ppm in C4 & CAM 2) reduces water loss

C4 and CAM photosynthesis reduces water loss C3 plants lose 500 -1000 H2O/CO2 fixed C4 plants lose 200 - 350 CAM plants lose 50 - 100

C4 photosynthesis = spatial isolation C4 plants have Kranz anatomy Mesophyll cells fix CO2 with PEP carboxylase Bundle sheath cells make CH20 by Calvin cycle