Bioenergetics-Quest for energy •  All cellular organisms need energy to grow and

survive. •  The energy derived from catabolism of growth

substrates is used to fuel anabolism (biosynthesis) and to fuel other cellular functions such as transport and motility.

•  Two catabolic modes –  Phototrophy- energy from light –  Chemotrophy - energy from chemical reactions

•  Chemotrophs catalyze thermodynamically favorable (exergonic) reactions and conserve part of the energy released, either as high energy ester bonds (such as ATP) or as an ion-motive force (usually protons).

•  Bacteria and Archaea are incredibly versatile at conserving energy– If there’s a buck to be made some bug will do it.....R. Wolfe (penny?)

Worth looking at

Gottschalk, 1985 2 in library Great chapter on fermentations

White, Drummond, and Fuqua 2012 Not in library - $110.67 at Amazon- 2006 Ed. much cheaper Lots of info – not very user friendly

Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41:100-180. Out of date, with nothing on chemiosmosis, but still a trove of information on fundamentals of anaerobic metabolism and killer tables at end

Free energy of reactions ∆G°' = ∑∆Gf° products - ∑∆Gf° reactants

-917.2 + 6(0) 6(-586.9) + 6(-39.9) glucose + 6O2 6HCO3- + 6H+

-917.2 -3760.8 ∆G°' = -3760.8 - (-917.2 ) = -2843.6 kJ/rxn (-237 kJ/e- pair)

glucose 2lactate- + 2H+

-917.2 2(-517.8) + 2 (-39.9) = -198.2 kJ/mol

4H2 + HCO3- + H+ CH4 + 3H2O

4(0) + (-586.9) +(-39.9) (-50.8) + 3(-237.2) = -135.6 kJ/rxn (-33.2 kJ/e- pair - but actually less)

Std conditions = 25 oC, 1 M solutes, 1 atm gases, aqueous (H20 ≈ 55 M) For ∆Go’, H+ is constant at 10-7 M (pH 7)

∆Gf values in Manual Appendix 3

Most from Thauer 1977; Bact. Rev (MMBR) 41:100-180

Balancing equations using H2

Example: Aerobic CH4 oxidation Hydrogenation Eqn 27- is backwards

Hydrogenation Eqn 43 – need to balance hydrogens

2O2 +4H2 4H2O -949.0 kJ/rxn

CH4+ 3H2O HCO3- + 4H2 + H+ +135.6 kJ/rxn

CH4+ 2O2 HCO3- + H+ + H2O -813.4 kJ/rxn

Methane + 3H2O HCO3- + 4H2 + H+ +135.6 kJ/rxn

Turn it around:

Concentration affects the free energy of reactions....

€

ΔG'= ΔGo '+RT ln (C)c (D)d

(A)a (B)b

For a reaction: aA + bB cC + dD

....particularly important for anaerobes €

ΔG'= ΔGo '+5.7log (C)c (D)d

(A)a (B)b

“Free energy form of the Nernst Equation”

At 25o C:

Effect of H2 partial pressure on methanogenesis

H2 typically 10-4-10-5 atm in methanogenic habitats

-150

-100

-50

0

50

10-7 10-6 10-5 10-4 10-3 10-2 10-1 100

∆G' (

kJ/rx

n)

H2 Partial Pressure (atm)

Effect of H2 partial pressure on ∆G’ of methanogenesis

slope = 22.8 (5.7 x 4) kJ per 10fold change

The electron tower

Substrate-level phosphorylation - ATP

∆Go’synthesis = +31.8 kJ/mol from ADP ≈ +40-80 kJ/mol under physiological conditions

Five ATPs are hydrolyzed to ADP to form each amide bond in a protein

R. Thauer MD 2010

Substrate-level phosphorylation: high-energy phosphoester compounds

Name Structure∆G°' of

hydrolysis(kJ/mol)

1,3-Diphosphoglycerate C C C OOH

O PO32-

H

H OH2-O3P

-52

Phosphoenol pyruvate H2C C COO–OPO32- -51

Acetyl phosphate H3C C OO

PO32- -45

Carbamyl phosphate H2N C OO

PO32- -40

Pyrophosphate2-O3P O PO3

2--24

Substrate-level phosphorylation: acyl coenzyme A thioesters

No resonance CH3-C–O

–

O –

Resonance

Acyl coenzyme A thioesters can be cashed in as ATP

Fatty acid or succinate as fermentation product Acyl CoA was probably cashed in as ATP

Fermentation •  Latin: "fermentum" - brewing beverages -

connotation of bubbling •  Alchemy: a process in which organic chemicals

were transformed –  Still used by industrial microbiologists, e.g. the "penicillin

fermentation” •  Early 20th century: metabolism of organics in the

absence of oxygen •  Brock (13th): Anaerobic catabolism of an organic

compound in which the compound serves both as electron donor and an electron acceptor and in which ATP is usually produced via substrate-level phosphorylation (SLP)

EMP pathway

Now easier to cleave

-ATP -ATP

+2ATP

+2ATP

Aldehyde high energy

+2NADH

Fermentation •  Another glycolysis pathway often used by microbes is the Entner

Douderoff (ED) pathway, which only conserves 1 ATP/glucose •  There are many variations on these pathways especially in Archaea

Selig et al. Arch Microbiol. 167:217 (1997)

Glycolysis diversity

Fermentation •  Net result of EMP is that glucose is converted to pyruvate with the

production of NADH, which needs to be re-oxidized •  In aerobes and some anaerobic respirers, the electrons can go

down the electron transport chain to the electron acceptor. •  Fermentative organisms don't have that option •  Must dispose of electrons from glycolysis •  Show three (and a half) solutions

Glucose

2 NADH + 2 H+2 ATP

2 ADP + 2Pi

2 Pyruvic acid

2 NAD+

The simplest solution

•  Dump electrons from NADH directly on pyruvic acid

•  Lactic acid is produced by hypoxic animal tissues and tumor cells.

•  Numerous microorganisms produce lactic acid

•  Most prominent are the "Lactic Acid Bacteria” in the Firmicutes

H3C CO

CO

OH

H3C COH

CO

OH

Pyruvicacid

Lacticacid

NADH + H+

NAD+

H

LactateDehydrogenase

The alcoholic fermentation: the second simplest solution

H3C C COOHO

H3C C HO

2 CO2

2 NADH + H+

2 NAD+

Pyruvic acid

Acetaldehyde

Alcohol dehydrogenase(ADH)

Pyruvate decarboxylase(PDC)

H3C C HOH

HEthanol

Glucose

2 NADH + 2 H+2 ATP

2 ADP + 2Pi 2 NAD+

2

2

2

Glucose --> 2 Ethanol + 2CO2Pathway in yeast

Also in Zymomonas mobilis, an Alphaproteobacterium Uses Entner Douderoff pathway and only gets 1 ATP/Glucose

Pyruvate-ferredoxin oxidoreductase: the clostridial solution

•  PFO and ferredoxin (Fd) are iron-sulfur (FeS) proteins

•  Pyruvate is a powerful reductant (pyruvate/acetate couple = -680 mv)

•  PFO can reduce Fd

•  Model of Fd showing two 4Fe4S groups

•  Eo’ of Fd ~ -400 mv •  Close to H2 (-414 mv)

•  Clostridia are often vigorous H2 producers

S – light yellow Fe – dark yellow

Hydrogenases •  Carry out the seemingly simple reaction: H2 2e– + 2H+

Ribbon model of [FeFe] hydrogenase showing FeS centers leading to active site

H2ase active sites

From: Science 321:572, 2008

The phosphoroclastic reaction H3C C COO-

O

H3C C S-CoAO

H3C C OPO32-

CO2

HS-CoA

O

Fdox

Fdred2H+

H3C C O-

HPO42-

H2

O

HS-CoA

ADP

+ ATP

Pyruvate

Acetyl-CoA

Acetyl-phosphate

Acetate

Acetate kinase

Phosphotransacetylase

Pyruvate:ferredoxinoxidoreductase Hydrogenase

Pyruvate + ADP + Pi <----> Acetate + H2 + CO2 + ATP

Thioclastic?

Fermentation of glucose units by C. thermocellum

Glucose + 3ADP + 3Pi --> Ethanol + Acetate + 2H2 + 2CO2 + 3ATP

H3C C COO-O

H3C C S-CoAO

H3C C OPO32-

O

H3C C O-O

2 HS-CoA

2 CO2

Fdox

Fdred4H+

H3C C S-CoAO

2H2PFO

Glucose

2NADH

H3C C HO

HPO42-

HS-CoA

2

H3C C HOH

+ ATPH

ADP

2 ATP

HS-CoAPTA

AK

AldehydeDehydrogenase

AlcoholDehydrogenase

The homoacetate “fermentation” by Moorella thermoacetica

Glucose ---> 3CH3COOH

Respiration?

Fermentation?

4 ATP “+” Drake and Daniel, Res. Microbiol. 155:869 (2004)

Butyrate fermentation in clostridia

From Gottschalk

Some Cl. ferment AAs Products include: putrescine, cadaverine, branched chain FAs, H2S, methyl mercaptan

Also butanol and acetone

The E. coli solution: pyruvate-formate lyase

H3C C S-CoAO

HS-CoA H3C C COO-

O

+ HCOOH

Formate-H2 lyase Membrane bound Resembles Complex I

H

H2 + CO2

H+

PFL Glycine radical at active site O2 labile

The E. coli mixed acid fermentation - a little bit of everything glucose

(H) ADP ATP

PEP ADP ATP

pyruvate

CO2 oxalo- acetate

TCA cycle

fumarate

succinate

Fumarate reductase

H+

formate

(100)

CO2 H2

Acetyl-CoA

CoA

Acetaldehyde

(H)

ethanol Acetyl-Pi

acetate

lactate

ADP ATP

Pi CoA

(H)

(H)

H+

(49.8)

(35.5)

(79.5)

(88.0) (75.0)

(2.4)

(10.7)

After Gottschalk, Bacterial Metabolism, 1985

= 600 mol C

Products = 531 mol C

Fermentations: summary •  O2 is limiting in many environments and organisms

need to dispose their electrons •  One solution is fermentation, using the organic

substrate as the electron acceptor •  Some facultative and aerotolerant anaerobes use

simple fermentations producing lactate or ethanol as products

•  Most true anaerobes increase their energetic yield by making acyl-CoA intermediates which can be cashed in as ATP

•  These pathways usually involve disposing of electrons as H2 in the phosphoroclastic reaction or PFL

A controversial proposal

Proton motive force

+++----+

Pump

A

B

H+H+

H+H+

H+

H+

H+

H+

H+H+H+

H+H+

H+

H+

H+ H+H+H+

H+ H+H+

H+H+

H+H+

H+H+

H+H+

H+

H+

H+

H+H+H+

H+

H+H+H+

H+H+

H+

H+

H+

H+

H+

Protons are pumped from compartment A to B. Two forces can drive them back into A 1)  the concentration difference

(∆pH) 2)  electrostatic attraction (∆Ψ).

The H+ concentration gradient component of the force can be expressed in volts as: RT/nF ln (H+

out)/(H+in)

= 0.059 log (H+out)/(H+

in) = -0.059 ∆pH The electrostatic force can be expressed in volts as: RT/nF ln (ionsout)/(ionsin) = 0.059 log (ionsout)/(ionsin) = ∆Ψ The total proton motive force (∆p) is: ∆p = ∆Ψ - 0.059 ∆pH

In an actively metabolizing cell, ∆p is typically 150-200 mv (0.15-0.2 v)

Rotary ion-pumping ATPases •  Three types: F1Fo (mitochondria, chloroplasts, and many bacteria),

A1Ao (mainly Archaea, some bacteria), V1Vo (acidify euk vacuoles) •  F1 in cytoplasm (matrix in mitos), Fo in membrane •  F1 – three alpha/beta dimers, each binds an (ADP + Pi/ATP) •  c subunits in Fo are proton channels, and along with the stalk

rotate (100X/sec) relative to the other subunits

Cytoplasm/matrix

Membrane

Outside/periplasm

F1

Fo

F1Fo in action Higher H+ outside (PMF)

Rotary ion-pumping ATPases •  ATPases are reversible – may help you to think of as an ATP-

powered proton pump (fan vs windmill) •  Each alpha/beta dimer in F1 converts 1 ADP + Pi to ATP per

360o rotation so there are 3 ATP altogether •  The question of how many H+/ATP (2,3,4?) was only settled

by a crystal structure of a yeast mitochondrion ATPase •  It had 10 c subunits so that per rotation there are 10/3 or 3.33

H+/ATP Science 286:1700 (1999)

ATPase predicted stoichiometries

Organism c subunits cation/ATP Beef heart mitochondria 8 2.67 Yeast mitochondria 10 3.33 Escherichia coli 10 3.33 Acetobacterium woodii 11 3.67 Propionigenium modestum 11 3.67 Thermus thermophilus 12 4 Spinach chloroplast 14 4.67 Various cyanobacteria 13-15 4.33-5 Methanopyrus kandleri 13 4.33

A Frankenstein-like reconstitution experiment •  In 1974 E. Racker at Cornell

teamed up with some German Halobacterium researchers to perform a multi-organism reconstruction experiment

•  Liposomes (membrane vesicles) from soybean lecithin

•  ATPase from beef-heart mitochondria

•  “Bacteriorhodopsin” from Halobacterium

•  ATPase and BR inserted "backward” into liposomes

•  When light was shined a proton circuit led to ATP production

E. coli expressing proteorhodopsin gene

Sodium circuit in Propionigenium modestum C C C C

H

H

H

H O–

OO

O–H+ + C C C

H

H

H

H O–

OH + CO2

Succinate Propionate

Proton motive force and rotary ATPases •  ∆p values in respiring organisms are typically 0.15 to 0.2 v, •  The production of ATP using 3.3 H+ is energetically

feasible –  ∆G= -nFE = 3.3 x 96.4 x (0.15-0.20) –  = –48-63 kJ/3.3 H+

•  ATPases with higher ratios don’t need as high ∆p –  for 15 subunits and 100 mv = 5 x 96.4 (0.10) = 48 kJ/ATP –  Like gears on a bike – but organisms can’t change

•  ATPases are reversible •  In fermentative heterotroph making ATP from SLP (e.g.

Streptococcus) –  Low ∆p and high ATP levels –  Needs ∆p for transport and motility –  ATPase hydrolyzes ATP to pump protons and provide a ∆p (or ∆Na+)

Electron transport-carriers

Couple E°' (v)

H+/H2 -0.41Ferredoxin (Clostridium) -0.41NAD(P)/NAD(P)H -0.32FMN/FMNH2 in NADH dehydrogenase -0.30FeS centers in NADH dehydrogenase -0.35 - 0.0Free FAD/FADH2 -0.22Free FMN/FMNH2 -0.19Menaquinone/menaquinol -0.07Ubiquinone/ubiquinol +0.13cytochrome b +0.06 - +0.26cytochrome c +0.25 - + 0.36Rieske iron sulfur protein +0.28cytochrome a +0.29cytochrome a3 +0.55O2/H2O +0.81

Properties of e- carriers at the end.

Moving protons via electron transport

Complex I – NADH/quinone oxidoreductase A proton pump

~95 Å

Quinone reduction site cytoplasm Q + 2e– + 2H+ QH2

Hydrophilic arm Cytoplasm ~280 kDa

Hydrophobic arm Membrane ~270 kDa

From Nature 465:441 (2010)

9 FeS centers 2e–

1e–

Periplasm

Electron transport in aerobically grown E. coli

NADH + H+ NAD+

QH2

QH2 QH2 Q

2H+

2e–

Q QH2

2H+ 4H+

4H+

b

CuB/o3

2e–

2H+ + 0.5O2 H2O

2H+

2H+ 8H+

8H+ 2.4 ADP + Pi 2.4 ATP

Out (periplasm)

In (cytoplasm)

Complex I Complex IV – quinol oxidase b and o3 are hemes CuB is a copper site

Electron transport pathways in E. coli

b CuB/o3

O + 2H+

H2O FMN FeS9

NADH + H+

NAD+

Q

4H+

2H+

NDH I – Complex I – Normal O2 Normal O2

bo3 quinol oxidase

NADH + H+

NAD+

FAD

NDH II – High O2

FMN FeS b

Fumarate + 2H+

Succinate Anaerobic

bd O + 2H+

H2O Low O2

bd quinol oxidase

Nitrate reductase

b FeS Mo

NO3– + 2H+

NO2– + H2O

Anaerobic + NO3–

2H+

FMN FeS b

Succinate

Fumarate + 2H+

Succinate DH – Complex II

Eo’ ≈ 0 v

Electron transport in aerobic Paracoccus denitrificans, an Alphaproteobacterium related to mitochondria

Rhodopseudomonas palustrisBradyrhizobium japonicum"Photorhizobium thompsonianum"

Nitrobacter winogradskyiMethylobacterium extorquens

Rhodopseudomonas viridisMethylosinus trichosporium

Rhodomicrobium vannieliiHyphomicrobium vulgare

Rhizobium lotiBrucella abortusBartonella bacilliformis

Rhizobium melilotiAgrobacterium tumefaciensRhizobium leguminosarumAgrobacterium rhizogenes

Caulobacter crescentusSphingomonas paucimobilis

Zymomonas mobilisErythrobacter longus

Rhodobacter capsulatusRhodobacter sphaeroidesParacoccus denitrificans

Zea mays - mitochondrionWolbachia pipientis

Rickettsia rickettsiiRickettsia prowazekii

Rhodospirillum rubrumMagnetospirillum magnetotacticumAzospirillum brasilense

Rhodopila globiformisAcetobacter aceti

Obligate intracellularparasites

(has respiratory system resembling mitochondria)

}

} PNS

Part of an Alphaproteobacteria tree

Electron bifurcation

Electron transport in the aerobic Archaeon Sulfolobus

•  It lacks complex I, uses a different quinone, and Complexes III and IV form a "supercomplex" without free cyt c,

•  Still, the electron transport chain is similar to those in Bacteria •  Was the ancestor of the Bacteria and Archaea an aerobe? Phylogenetic

trees for the large subunit of copper oxidases are inconclusive

Other Archaea have Complex I

A truncated electron transport chain in Acidithiobacillus ferrooxidans

• The lithotroph A. ferrooxidans (formerly Thiobacillus) grows aerobically at pH 2 by oxidizing Fe2+ to Fe3+

• Fe2+ is not a strong enough reductant (+0.65 v at pH 2) to reduce NAD+ or quinones

• The electrons feed into the terminal oxidase through two high potential carriers (rusticyanin and cytc533) in the periplasm

Fe2+ Fe3+

cyt aa3

O + 2H+ H2O

2H+

Cyc2? Outer Membrane

Inner Membrane

Periplasm RC cytc553

Cell gaining ATP from SLP using its ATPase to generate a ∆p

Summary H+

NADH

Glucose

2 Pyruvate-

6 CO2

I IIIQ

c

H+ H+ H+

O2 H2O

IV

H+

ADP +PiATP

Periplasm

Membrane

SoluteBiosynthesistransport, etc.

Cytoplasm

F1Fo ATPase

Transporter

Respiratory cell with ET chain resembling that in Paracoccus/mitochondria

Volta 2013

Electron transport •  Organisms transport electrons through a

chain of carriers, going energetically downhill from an electron donor to the final electron acceptor

•  Some carriers carry only electrons, while others carry an electron plus a proton, the equivalent of an H atom

•  Organisms can take advantage of this to develop a ∆p across the cell membrane

Electron donorox

Electron donorred

Carrier 1red

Carrier 1ox

Carrier 2ox

Carrier 2red Electron acceptorox

Electron acceptorred

Nicotinamide adenine dinucleotide (NAD+)

Carries 1 H+ + 2e- (hydride)

Eo' = -0.32 v (-320 mv)

Flavins - flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)

Carry one or two H (H+ + e-) Eo' = -0.2 v (-200 mv) Usually serve as prosthetic groups in proteins (flavoproteins) Eo' in proteins can be as low as -0.4 v (flavodoxins) or as high as 0 v

Quinones O

O

CH3H3CO

H3CO

OH

OR

CH3H3CO

H3CO

OH

OHR

CH3H3CO

H3CO

O

OR

CH3

O

OR'

SCH3

S

O

O

.

H+ + e-

R

HH3C

H3C

H+ + e-

H+ + e- H+ + e-Ubiquinone

4-8

Semi-quinone radical

Ubiquinol Plastiquinone

Menaquinone

"Calderiellaquinone"

• Have long hydrocarbon chain that anchors them to the membrane

• Carry one or two H (H+ + e-)

• Ubiquinone commonly found in aerobes, Eo' = +0.13 v

• Menaquinone more common in anaerobes, Eo' = -0.07 v

• Plastiquinone found in chloroplasts and cyanobacteria, Eo' = 0 v

• Calderiellaquinone is found in Sulfolobus, a sulfur-oxidizing member of the Crenarchaeota, Eo' = +0.1 v

Cytochromes

N N

N N

CH2 CH2

CH3H3C

HC

CH3 HC

CH3

H2C

COO–

H2C

COO–

CH2

H2C

Fe

N N

N N

CH2 CH2

CH3H3C

CH

CH3 CH

CH3

H2C

COO–

H2C

COO–

CH3

cys-S

H3C

cys-S

Fe

Heme b Heme c showing covalent links to protein

• Proteins containing heme prosthetic groups • Fe complexed in various tetrapyrrole rings • Can contain hemes a, b, d, o, or c (covalently linked) • Carry a single electron reducing Fe3+ to Fe2+ • Eo' of different cytochromes can vary greatly from -0.3 to + 0.6 v

Iron-sulfur proteins

2Fe/2S cluster

Rieske 2Fe/2S cluster

4Fe/4S cluster

• Iron sulfur (FeS) proteins have FeS clusters as electron carrying prosthetic groups

• Each cluster can carry a single electron (reducing an Fe3+ to Fe2+) and FeS proteins can have more than one FeS group

• FeS clusters are usually liganded by sulfur groups of cysteines except in the "Rieske" proteins, in which two of the ligands are Ns in histidine

•  The Eo' for FeS proteins is typically reducing (-0.53 to 0) except for the Rieske type with a potential of + 0.28

• The importance of FeS proteins was not appreciated because their light/UV spectrum doesn't change significantly on reduction. Can use EPR to detect.

• FeS proteins are probably ancient, derived from naturally forming FeS precipitates