Course ProjectEngineering electricity production by living organismshttp://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 speciesIn 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 makeH2 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 PSIEach e- drives cyt b6/fUse PMF to make ATPPSII replaces e- from H2O forming O2

Z-scheme energetics

Physical organization of Z-schemePS 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-schemePSII 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 Photochemistry1) LHCII absorbs a photon2) energy is transferred to P680

PSII Photochemistry3) P680* reduces pheophytin ( chl a with 2 H+ instead of Mg2+) = primary electron acceptor

PSII Photochemistry3) P680* reduces pheophytin ( chl a with 2 H+ instead of Mg2+) = primary electron acceptorcharge separation traps the electron

PSII Photochemistry4) pheophytin reduces PQA (plastoquinone bound to D2)moves electron away fromP680+ & closer to stroma

PSII Photochemistry

5) PQA reduces PQB (forms PQB- )

PSII Photochemistry6) P680+ acquires another electron , and steps 1-4 are repeated

PSII Photochemistry7) PQA reduces PQB - -> forms PQB2-

PSII Photochemistry8) PQB2- acquires 2 H+ from stroma forms PQH2 (and adds to ∆pH)

PSII Photochemistry9) PQH2 diffuses within bilayer to cyt b6/f- is replaced within D1 by an oxidized PQ

Photolysis: Making Oxygen1) P680+ oxidizes tyrZ ( an amino acid of protein D1)

Photolysis: Making Oxygen2) tyrZ + oxidizes one of the Mn atoms in the OECMn cluster is an e- reservoir

Photolysis: Making Oxygen2) tyrZ + oxidizes one of the Mn atoms in the OECMn cluster is an e- reservoirOnce 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/O2Mn cluster cycles S0 -> S4

Reset to S0 by taking 4 e-

from 2 H2O

Electron transport from PSII to PSI1) PQH2 diffuses to cyt b6/f2) 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 PSI3) Fe/S reduces plastocyanin via cyt fcyt b6 reduces PQ to form PQ-

Electron transport from PSII to PSI4) repeat process, Fe/S reduces plastocyanin via cyt fcyt 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 Ferredoxin1) LHCI absorbs a photon2) P700* reduces A03) 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 reduceNADP reductaseNADP reductase reduces NADP+

Electron transport from Ferredoxin to NADP+2 Ferredoxin reduceNADP reductaseNADP 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 synthesisPMF mainly due to ∆pHis used to make ATP-> very little membrane potential, due to transport of other ionsthylakoid lumen pH is < 5 cf stroma pH is 8pH is made by ETS, cyclic photophosphorylation,watersplitting & NADPH synth

Chemiosmotic ATP synthesisStructure of ATP synthaseCF1 head: exposed to stromaCF0 base: Integral membrane protein

a & b2 subunits form stator that immobilizes & F1 subunitsa is also an H+ channelc subunits rotate as H+

pass through & also rotatec, & form a rotor

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

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

ADP + Pi bind to subunitthen spontaneously form ATP

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

ADP + Pi bind to subunitthen spontaneously form ATP

∆G for ADP + Pi = ATP is ~0role of H+ translocation is toforce enzyme to release ATP!

Binding Change mechanism of ATP synthesis1) H+ translocation alters affinity of active site for ATP2) 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 synthesis1) H+ translocation alters affinity of active site for ATP2) Each active site ratchets through 3 conformations that have different affinities for ATP, ADP & Pi

3) ATP is synthesized by rotational catalysisg subunit rotates as H+ pass through Fo, forces each active site to sequentially adopt the 3 conformations

Evidence supporting chemiosmosisRacker & Stoeckenius (1974) reconstituted bacteriorhodopsin and ATP synthase in liposomesBacteriorhodopsin uses light to pump H+

make ATP onlyin the light

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

Engineering the light reactions?

Engineering the light reactions?1.Absorb full spectrum

Engineering the light reactions?1.Absorb full spectrum2.Improve efficiency of transport w/in PSI & PSII (or recover the energy)

Engineering the light reactions?1.Absorb full spectrum2.Improve efficiency of transport w/in PSI & PSII (or recover the energy)3.Transport electrons to another acceptor•From OEE•From PQH2

•From Fd

Engineering the light reactions?1.Absorb full spectrum2.Improve efficiency of transport w/in PSI & PSII (or recover the energy)3.Transport electrons to another acceptor•From OEE•From PQH2

•From Fd: use hydrogenase to make H2

Engineering the light reactions?1.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?1.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) reactionsThe Calvin cycle

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

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

Light-independent (dark) reactions1) fixing CO2

2) reversing glycolysis3) regenerating RuBP

fixing CO2

1) RuBP binds CO2

fixing CO2

1) RuBP binds CO2 2) rapidly splits into two 3-Phosphoglycerate

• therefore called C3 photosynthesis

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate

• therefore called C3 photosynthesis•detected by immediately killing cells fed 14CO2

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase)the most important & abundant protein on earth

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase)the most important & abundant protein on earth

•Lousy Km

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase)the most important & abundant protein on earth

•Lousy Km

•Rotten Vmax!

Reversing glycolysisconverts 3-Phosphoglycerate to G3Pconsumes 1 ATP & 1 NADPH

Reversing glycolysisG3P has 2 possible fates

1) 1 in 6 becomes (CH2O)n

Reversing glycolysisG3P has 2 possible fates

1) 1 in 6 becomes (CH2O)n

2) 5 in 6 regenerate RuBP

Reversing glycolysis1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell)

Reversing glycolysis1 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 glycolysis1 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 sucrosePi/triosePO4

antiporter onlytrades DHAP for Pi

Reversing glycolysis1 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 sucrosePi/triosePO4

antiporter onlytrades DHAP for Pimechanism to regulate PS

Regenerating RuBPG3P has 2 possible fates

5 in 6 regenerate RuBPnecessary to keep cycle going

Regenerating RuBPBasic problem: converting a 3C to a 5C compoundfeed in five 3C sugars, recover three 5C sugars

Regenerating RuBPBasic problem: converting a 3C to a 5C compoundmust assemble intermediates that can be broken into 5 C sugars after adding 3C subunit

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C4C + 3C = 7C

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C4C + 3C = 7C7C + 3C = 5C + 5C

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C4C + 3C = 7C7C + 3C = 5C + 5CUses 1 ATP/RuBP

Light-independent (dark) reactionsbuild up pools of intermediates , occasionally remove onevery complicated book-keeping

Light-independent (dark) reactionsbuild up pools of intermediates , occasionally remove onevery complicated book-keepingUse 12 NADPH and 18 ATP to make one 6C sugar

Regulating the Calvin CycleRubisco is main rate-limiting step

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways 1) Rubisco activase : uses ATP to activate rubisco

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pH: rubisco is most active at pH > 8 (in dark pH is ~7.2)

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pHb) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pHb) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in darkMg2+ moves from thylakoid lumen to stroma to maintain charge neutrality

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pHb) [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 CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) 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 CycleSeveral 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 CycleSeveral Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin

contain disulfide bonds which get oxidized in the darkin light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme

S - S

2Fdox

2Fdred

PSI +

PSII

light

2e-

oxidizedthioredoxin

reducedthioredoxin

S - S

SH SHoxidizedenzyme

(inactive)

reducedenzyme(active)

SH SH

Regulating the Calvin CycleSeveral Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin

contain disulfide bonds which get oxidized in the darkin light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzymeHow light reactions talk to the Calvin cycle

S - S

2Fdox

2Fdred

PSI +

PSII

light

2e-

oxidizedthioredoxin

reducedthioredoxin

S - S

SH SHoxidizedenzyme

(inactive)

reducedenzyme(active)

SH SH

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

RuBP + O2 <=> 3-phosphoglycerate + PhosphoglycolateReleases CO2 without making ATP or NADH

PHOTORESPIRATION Releases CO2 without making ATP or NADH

Called photorespiration : undoes photosynthesis

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

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

RuBP + O2 <=> 3-phosphoglycerate + PhosphoglycolateC3 plants can lose 25%-50% of their fixed carbonBoth rxns occur at same active site

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

Detoxifying Glycolate1) glycolate is shuttled to peroxisomes

Detoxifying Glycolate1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine

produce H2O2

Detoxifying Glycolate1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine3) glycine is sent to mitochondria

Detoxifying Glycolate1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine3) glycine is sent to mitochondria4) mitochondria convert 2 glycine to 1 serine + 1 CO2

Why photorespiration loses CO2

Detoxifying Glycolate1) glycolate is shuttled to peroxisomes2) peroxisomes convert it to glycine3) glycine is sent to mitochondria4) mitochondria convert 2 glycine to 1 serine + 1 CO2

5) serine is returned to peroxisome

Detoxifying Glycolate1) glycolate is shuttled to peroxisomes2) peroxisomes convert it to glycine3) glycine is sent to mitochondria4) mitochondria convert 2 glycine to 1 serine + 1 CO2

5) serine is returned to peroxisome6) peroxisome converts it to glycerate & returns it to chloroplast

Detoxifying GlycolateWhy peroxisomes are next to cp and mito in C3 plants

Mitochondrion

C4 and CAM photosynthesisRubisco can use O2 as substrate instead of CO2

[CO2] is 1/600 [O2] _-> usually discriminate well

C4 and CAM photosynthesisRubisco can use O2 as substrate instead of CO2

[CO2] is 1/600 [O2]

Photorespiration increases with temperature

C4 and CAM photosynthesisRubisco can use O2 as substrate instead of CO2

[CO2] is 1/600 [O2]

Photorespiration increases with temperatureSolution: increase [CO2] at rubisco

C4 and CAM photosynthesisSolution: increase [CO2] at rubiscoC4 & CAM = adaptations that reduce PR & water loss

C4 and CAM photosynthesisAdaptations that reduce PR & water lossBoth fix CO2 with a different enzyme

C4 and CAM photosynthesisAdaptations that reduce PR & water lossBoth fix CO2 with a different enzymelater release CO2 to be fixed by rubiscouse energy to increase [CO2] at rubisco

C4 and CAM photosynthesisAdaptations that reduce PR & water lossBoth fix CO2 with a different enzymelater release CO2 to be fixed by rubisco

use energy to increase [CO2] at rubiscoC4 isolates rubisco spatially (e.g. corn)

C4 and CAM photosynthesisAdaptations that reduce PR & water lossBoth fix CO2 with a different enzymelater release CO2 to be fixed by rubiscouse energy to increase [CO2] at rubiscoC4 isolates rubisco spatially (e.g. corn)CAM isolates rubisco temporally (e.g. cacti)

C4 and CAM photosynthesisC4 isolates rubisco spatially (e.g. corn)CAM isolates rubisco temporally (e.g. cacti)Advantages: 1) increases [CO2] at rubisco

C4 and CAM photosynthesisAdvantages: 1) increases [CO2] at rubisco

reduces PRprevents CO2 from escaping

C4 and CAM photosynthesisAdvantages: 1) increases [CO2] at rubisco

reduces PRCO2 compensation point is 20-100 ppm in C3 0-5 ppm in C4 & CAM

C4 and CAM photosynthesisAdvantages: 1) increases [CO2] at rubisco

reduces PRCO2 compensation point is 20-100 ppm in C3 0-5 ppm in C4 & CAM

2) reduces water loss

C4 and CAM photosynthesisreduces water loss

C3 plants lose 500 -1000 H2O/CO2 fixedC4 plants lose 200 - 350CAM plants lose 50 - 100

C4 photosynthesis = spatial isolationC4 plants have Kranz anatomyMesophyll cells fix CO2 with PEP carboxylase

Bundle sheath cells make CH20 by Calvin cycle