Bioenergetics: Lecture of May 21, 2009

Introduction to bioenergetics.

The thermodynamics of biological energy production

Kinetic aspects of bioenergetic processes

The molecular and cellular organization of bioenergetic systems

Photosynthesis

structure of the photosynthetic reaction center photosynthetic electron transfer plant photosystems

plant photosystemsCO2 fixation photosynthetic efficiency

Biofuels Biofuels

Ethanol

Respiration and ATP synthesis

Haber-Bosch process and biological nitrogen fixation

photosynthesis - the reaction center h

Bchl2

membrane

2

Bchl

QA QB

Bph

A QB

Fe

Bchl -chlorophyll

Bph - bacteriopheophytin

Q - quinone

why is the back reaction (to (Bchl)2+) so unfavorable?

why is the B branch so much slower than the A branch?

the inverted region

organization of photosynthetic electron transfer system

cytochrome c h

H+

membraneQ

QH2

photosynthetic

H+

p y

reaction center

cytochrome bc1

Quinone structures and redox propertiesQuinone structures and redox properties

• lipid soluble carrier of electrons and protons

O

R1

O•

R

OH

RR1

R2 R3

R1

R2 R

R1

R2 R

+e- +e- + 2H+

O

R2 R3

oxidized quinone

O-

R2 R3

OH

R2 R3

semiquinone hydroquinoneoxidized quinone(Q10)

semiquinone(can be protonated)

y q

Q l idi d d i i t tQA - only oxidized and semiquinone states;

QB - undergoes all three redox states

some classes of herbicides are quinone analogs that bind tightly toquinone analogs that bind tightly to QB site, but are not reduced.

how do the quinones get out?

Roszak et al. Science 302, 1969 (2003)

Replenishing electrons to RC+

the cytochrome c - RC complexthe cytochrome c RC complex

PDB ID 1L9J

complementary interactions assure specificity in interprotein electron transfer

cyt c2p y p

RC

QH2 oxidation in cytochrome bc1: the Q-cycle

Overall stoichiometry of cyclic electron transfer in y yphotosynthetic bacteria:

• the RC takes up one H+ from the cytoplasm per electron• the RC takes up one H+ from the cytoplasm per electron

• in cytochrome bc1, one H+ is taken up from the t l b t t l d t th i lcytoplasm, but two are released to the periplasm,

per electron.

net: 2H+ are translocated across the membrane per electron;

this creates a ∆p that is used to synthesize ATP this creates a ∆p that is used to synthesize ATP,

but cannot generate reducing equivalents for biosynthesis in a cyclic processy y p

photosynthesis in plants

H+

Buchanan, Gruissem, Jones

Biochemistry and Molecular Biology of Plants

plant photosystems (I and II)

http://www.bio.ic.ac.uk/research/barber/photosystemII.html

Z-scheme for non-cyclic photosynthesis electron transfer

photosystem II

(plant photosystems are depicted “upside-down” relative to bacterial RCs…)

photosystem IIZouni et al Nature 409, 739 (2001)

PDB ID 1FE1

Ferreira, Iverson et al. Science 303, 1831 (2004)

Cofactor arrangement in PSII

P680 th ti f hl h ll i i di i h tP680 - the separation of chlorophyll rings in dimer is somewhat greater than in the RC; shows more monomeric character

Light harvesting antenna system in PSII

oxygen evolution

3

oxygen evolution during photosynthesis

steady state

O2 evolving state

dark adapted state

Buchanan, Gruissem, Jones

Biochemistry and Molecular Biology of Plants

Oxygen evolving center:tetra-manganese cluster

Z i t lGuskov et al

Ferreira, Iverson et al.Science 303, 1831 (2004)

Zouni et al Nature 409, 739 (2001)

Nature Structural Biology 16, 334 (2009)

Photosystem II contains several lipid molecules within the structure

Lipids are probably important for turnover of D1 polypeptide, which is recycled

every 30 min in the celly

RC core

photosystem I p yJordan et al. , Nature 411, 909 (2000)

PDB ID: 1JB0

organization of electron transfer centers in PSI

P700 di f hl h llP700 - dimer of chlorophylls

arrangement of antennae

chlorophylls in PS Ic o op y s S

Dutton et al. Adv. Prot. Chem. 63 (2003)

RubiscoRubisco

Reactions Catalyzed by Rubisco

All Rubisco Enzymes are not specific for CO2 over O2

Many RuBisCo-like enzymes sequencesidentified in soil bacteria and archaea: functions are unknownfunctions are unknown

CO2 metabolism - needs to exchange between gas and solution phases:

CO2 (gas) CO2 (aq) + H2O H2CO3 HCO3- + H+

CO2 (gas) CO2 (aq) ∆G˚’ = + 8.4 kJ/moleCO2 (gas) CO2 (aq) ∆G 8.4 kJ/mole

CO2 (aq) + H2O HCO3- + H+ ∆G˚’ = + 4.7 kJ/mole

reversible hydration catalyzed by carbonic anhydrase

M. thermoautotrophicum CAM. thermophila carbonic anhydrase

Biofuels• Engineer cell metabolic pathways to produce

BiofuelsEngineer cell metabolic pathways to produce alcohol: methanol, ethanol, propanol, butanol

• Cells need fuel/energy: best source is glucosegy g• Cellulose can be degraded to glucose by

cellulases

Degradation of Cellulose is the Rate limiting stepRate limiting step

• Faster Cellulases• More stable cellulases (last longer in

incubator, and thus the total yield of degraded cellulase is greater)

• Microorganisms make cellulosomes which have l diff f ll l d diseveral different types of cellulose degrading

enzymes: Progressive degradation

Cellulases are processive enzymesCellulases are processive enzymes

Cl t idi th llClostridium thermocellum

Corn Stover ParticlesPre-treatment: 190C for 15 min

Pre-treatment changes susceptibility of cellulose to enzyme degradation

Utilization of forestland and agricultural land could produce 1.3 billion tons of pdry biomass

This would produce sufficient biofuels to displace 30 percent of domesticto displace 30 percent of domestic petroleum consumption

Ethanol Butanol GasolineEnergy content 84,000 110,000 115,000gyBTU/gal.

, , ,

Vapor pressure psi at 100F

2 0.33 4.5

Automobile compatibility

Max 10% 100% 100%

Biofuels

Biofuels will be a big part of America’s and the world’s energy futureBiofuels will be a big part of America s and the world s energy future.

Biofuels Initiative:•To make cellulosic ethanol cost competitive with gasoline by 2012. •To replace 30 percent of current levels of gasoline consumption with biofuels by 2030 (or 30x30).

•Increased economic growthEconomic opportunities for domestic, rural economies Decreased petroleum trade deficit

•Broad-based environmental benefitsReduced greenhouse gas emissions Reduced petroleum use in fuel production

•Improved national energy securityImproved national energy securityReduced reliance on foreign sources of energy Decreased threat of supply disruptions due to natural disasters, political instability, and price volatility

Metabolic EngineeringMetabolic Engineering

• Maximize the level of NAD availableMaximize the level of NAD available• Cell has to tolerate working concentrations

of substrate and productsof substrate and products

Engineered biochemical pathway to make alcohols from intermediates of the amino acid biosynthesis pathways

Atsumi, Liao Nature (2008) 351: 86-90

Engineered biochemical pathway to make alcohols from intermediates of the amino acid biosynthesis pathways

Atsumi, Liao Nature (2008) 351: 86-90

glucose 2 pyruvate Net reaction:

Engineering Alcohol Dehydrogenase Cofactor Specificity for Isobutanol Production

glucose 2 pyruvate

acetolactate

alsS2 NADH

CO2

Net reaction:

1 glucose + 2 NADPH + 2 NAD+

2 3 dihydroxy isovalerate

acetolactate

ilvCNADPH 1 isobutanol + 2 NADP+ + 2 NADH + 2 CO2

2,3-dihydroxy-isovalerate

keto isovalerate

ilvD

• 66% of glucose carbon results in isobutanol

• 33% carbon lost to CO-keto-isovalerate

isobutyraldehyde

kivDCO2

• 33% carbon lost to CO2

• As constructed the pathway has a co-factor imbalance

EhrlichPathway(Isobutanol)

isobutyraldehyde

i b t l

yqhDNADPH • To produce NADPH the cells have to

spend energy/carbon resulting in lower yield( ) isobutanol yield.

Changing the cofactor specificity of ilvC and yqhD would solve the imbalance.

Metabolic EngineeringMetabolic Engineering

• Maximize the level of NAD availableMaximize the level of NAD available• Cell has tolerate working concentrations of

substrate and productssubstrate and products

Project GoalsProject Goals• Engineer NADH-dependent KARI (ilvC) and IDH (yqhD) enzymes.

D li NADH d d IDH i h 0% f hD NADPH• Deliver a NADH-dependent IDH with 50% of yqhD NADPH-dependent isobutyraldehyde reduction activity by March 2009

• Integrate the engineered enzymes into the isobutanol pathway• Integrate the engineered enzymes into the isobutanol pathway.

• Optimize isobutanol production via strain development.

Clostridium thermocellumClostridium thermocellum– Can completely consume insoluble cellulose

Produces ethanol– Produces ethanol

Produces other products in addition to ethanol– Produces other products in addition to ethanol– Cannot genetically modify Clostridium

Multi-stage biofuel productionMulti stage biofuel production

http://www.ornl.gov/

Evolution of improved endoglucanases

ll l

•Cellulosomal cellulases from anaerobic bacteria Clostridium thermocellum will be parents of recombinant endoglucanases

cellulosomes

thermocellum will be parents of recombinant endoglucanases (EGs)

•Successful optimization of EG and CBH activity enablesSuccessful optimization of EG and CBH activity enables growth on cellulose

Thermostable Cellulases from SCHEMASCHEMA

MTIKEMPQPKTFGELKNL...KETSPIPQPKTFGPLGNL...KQASAIPQPKTYGPLKNL...WRRRGIPGPLGYPLVGSF...WIRKGVKGPRGLPFLGVI...FIRKGIKGPRGFPGIGML...WIRKGVKGPRGFPFFGVIWIRKGVKGPRGFPFFGVI...WIRKGVKGPRGFPFFGVI...WMRKGIKGPRGLPFFGII...WMRKGVKGPRGRPFVGVL...WRRRGVVGPMGFPVLGVF...REKIGLSGPEPHWFLGNL...REKIGLTGPEPHWFMGNL...RSSIGIPGPPVHWLWGNL...KVSKYPKGPLPLPFIGNI E =C KVSKYPKGPLPLPFIGNI...... E =Cijij

300

400

500

600C therophilumH insolensT reeseiHJPlus

Parent 2 Parent 3

Parent 1/u

gE

nzym

e

0

100

200

300ug

Glu

cose

/

50 55 60 65 70 75

Temperature (C)

Directed Evolution of Cyt P450 BM3 to convert propane to propanolconvert propane to propanol

Sugar polymer feedstocksSugar polymer feedstocks

Cellulase synthase can be d i b iexpressed in cyanobacteria

• Cellulase synthaseCellulase synthase can make non-crystalline cellulose

• Appears to also produce glucose in the media(?)