M3 - Membrane Bioenergetics and Electron Transport

Membrane Bioenergetics

How E is stored and used for biological purposes

Ways to Make ATP

1 Substrate Lvl phosphorylation

2 Oxidative Phosphorylation

Substrate Level phosphorylation

METABOLISM of high energy molecules in cytosol are COUPLED to transfer Pi to ADP to make ATP

Oxidative Phosphorylation

LIGHT or CHEMICAL energy are converted to electrochemical gradients in membrane that are COUPPLED to transfer Pi to ADP with ATP synthase.

Chemiosmotic Theory

Theory that electrochemical gradient can be FORMED BY ENZYMES on a membrane to do biological work

Electrochemical Gradient

Sodium or proton motive force --> Δp (or ΔμH+)

Membrane Associated enzymes

Enzymes that form chemical gradient at exposure to chemical or light energy

Δp = Δψ - 60ΔpH

Equation that represents the potential of a membrane to do work.

Δp

proton motive force (what's used to do work)

-> the more negative, the greater the potential to do work

Δψ

Membrane potential

Δψ= - 60 log [X+ in]/[X+ out]

Membrane potential equation

ΔpH

pH Gradient

ΔpH = pH in - pH out

pH Gradient equation

Steps in electrochemical gradient

1 membrane potential develops ( Δψ)

2 pH Gradient (ΔpH) develops

3 As pH Gradient (ΔpH) develops, positive ions pass through membrane

4 Membrane potential (Δψ) develops as e- pass across membrane

Ways to use electrochemical gradient to do work (4)

1 Antiport

2 ATP synthase

3 Flagellum Rotation

4 Solute uptake (simport)

antiport

Uses electrochemical gradient to transport one type of substance into cell while transporting out another type of substance (substances of same charge)

ATP synthase

Uses electrochemical gradient to turn/use this membrane bound protein to add Pi to ADP+

Solute uptake

Uses electrochemical gradient to uptake solutes into the cell.

EX: symport

Symport

Uses electrochemical gradient to transport two substances in the same direction across the membrane.

Reverse Electron Transport

Uses electrochemical gradient to do generate NADP. Causes e- to move backwards through gradient

-High concentrations of H+ or Na+ required to offset make E0 negative enough to move e-

-endergonic

-heavily reduced Q

-common in chemolithotrophs

ATP Synthase domains

F0 and F1 domains

F0 domain

Domain that harbors proton channel that spans the cell membrane

F1 domain

Domain that is on the inner membrane and harbors active site. Drives ATP synthesis with Δp or uses ATP hydrolysis to generate Δp

Types of ATP synthase

F1F0

A1A0

A1A0

F1F0 ATP synthase

Most common type of ATP synthase. Common in B, mitochondria, chloroplasts, some A. Is reversable and uses H+ (Na+ in anaerobic B)

V1V0 ATP synthase

ATP synthase found in vacuoles. NOT reversable (only ATP hydrolysis = ATPase). Uses H+ as e- acceptor (N+ in anaerobic B)

A1A0 ATP synthase

ATP synthase used in many A and some B. is reversable and uses Na+ and H+ as e- acceptors (H+ in methanogens).

-differs in structure; central stalk has 2 peripheral stalks

ATP synthase C-Subunit (F0 domain*)

Subunits in ATP synthase that are the part that rotates and binds H+. # ATP produced/stoichiometry varies with organism

-For every 360 rotation, produces 3 ATP in Yeast

-Large C likely selected for in phototrophs

Chemical Mechanisms to generating Electrochemical Gradients

1 ATP synthase

2 respiration

3 Methyl Transfer

4 Decarboxylation

5 End-Product Efflux

Light Mechanisms to generating Electrochemical Gradients

1 Photosynthesis

2 Photocycle

Photosynthesis

Oxidation of chlorophyl in reaction center generates Electrochemical Gradient

Photocycle

Conformational change of rhodopsin with membrane bound enzymes generates Electrochemical Gradient

ATP Hydrolysis

Common in organisms under fermentation conditions. ATP is converted to ADP & phosphate energized myosin heads (removal of phosphate) need energy.

-> substrate lvl phosphorylation dominates metabolism

-> how aerobes usually generate ATP***

Rhodopsin-Based Phototrophy and Photocycle

Predominant in ocean/aquatic/high saline envo

-> light-driven H+ pump generates Electrochemical Gradient

Chromophore (retinal)

light-sensitive group that captures light photons for photocycle.

-Forms schiff's base with lysine of rhodopsin

-Retinal covalently bonds

-Undergoes Cis -> Trans change w/ light

-transports H+ across cell to generate gradient

Methyltransferase

Enzyme common in methanogens and homoacetogens. Used in Wood-Ljngdahl pathway to generate Electrochemical Gradient

Decarboxylation

Uses CO2 to generate electrochemical gradient. 2 types

1 Membrane Bound

2 Soluble (Membrane Bound Antiporter)

Membrane bound decarboxylation

Decarboxylation common in Lactic Acid Bacteria and Enterobacter and w/ inside-out vesicles

1 decarboxylate generates ΔμNa+

2 ΔμNa+ converted to PMF with antiporter

3 ΔμNa+ syth ATP (w/ ATP synthase ofc)

4 ΔμNa+/H+ solute uptake w/ symport

Soluble Decarboxylation (Membrane-Bound Antiporter)

Decarboxylation of acid and H+ to monocarboxylic acid and CO2. Consumes [H+] in cell and antiporter reduces (-) charge out of cell

Oxalobacter formigenes

Organism that converts oxalate to formate with HP+ gradient (3 molc oxalate consumed per ATP synthesized)

End Product Flux

Concentration of organic metabolic end products can DRIVE SYMPORT w/ H+/Na+ & formation of electrochemical gradient

Respiratory Chains

Respiration uses membrane bound enzymes to cat RedOx RXNs and coupling with Na+/H+ to generate electrochemical gradient

terminal e- acceptors

anaerobes -> non-O2

aerobes -> O2

redox potential (E0)

Tendency of systems to couple to accept and donate e-. Determined by mixing equimolar ratios of two couples (Aox/Ared & Box/Bred).

-More positive = more potential

Steps in Respiration

1 Membrane bound enzymes cat RedOx RXN

2 Redox RXNs alternate between H+ and e- carriers

3 redox potential (E0) of the primary e- donor is < terminal e- acceptor

PMF of Neutrophiles

(live in pH 7-8.5). Anaerobic respiration doesn't involve reparatory chain, 3* ATP is used to maintain gradient

PMF of Acidophiles

(pH 1-4). Δψ is inverted so ATP must be hydrolyzed to counter negative charge on inside* of membrane. Work hard to maintain basic internal environment (is super favorable e- gradient)

PMF of Alkalophiles

(pH NOT favorable). So, Δψ is even more important. Na+ dependent for growth --> Na+/H+ antiporter needed to bring H+ inside cell to maintain neutral cytosol

PMF uncoupled at pH< pKa of acid

WEAK acids mess up cells bc their pKA is lowkey between the environmental pH and the pH in the cell, meaning they can diffuse across cell membranes and mess stuff up

H+ Carriers

Flavins and Quinones

e- Carriers

Fe-S clusters, cytochromes

Flavin bound proteins

Proteins that bind FLAVIN groups (FMN, FAD) derived from riboflavin (V B12). are COENZYMES that carry 2H (2H & 2e-)

EX: NADH dehydrogenase, succinate dehydrogenase (SDH, complex II)

NADH dehydrogenase (Complex 1)

FAD

Flavin protein that is predominantly bound non-covalently to flavoproteins

Quinones

Molecules that SHUTTLE 2H between non-mobile enzyme complexes. Bind non-cov and differ in redox potential. V-K derived lipophilic 2H carriers with isoprene [6-10 units] side chains

Ex: ubiquinone (UQ, Q, CoQ), menaquinone (MQ, MK), plastoquinone (PQ)

Cytochromes

ONE e- carriers that bind to heme mostly non-cov

Fe-S Proteins

Made of non-heme Iron, Acid liable S2-, and are ligated protein by Cys side chain.

-E0 ranges from -400 to 350

Ex: Fe2S2, Fe4S4, Fe3S4

Mechanisms of H+ translocation (3,2)

1 H-Pump

2 Q-Loop

3 Q-Cycle

A Vectorial

B Scalar

Scalar

Type of H+ translocation where Q-loop is reduced on cytosolic side. QH2 travels w/in membrane and os oxidized on external face of membrane which releases H+

Vectorial

Type of H+ translocation where e- is transferred directly to coupled H+

H-Pump

Vectorial, 2H+/2e-, syt oxidase complex IV

-H+ translocation that has 2 distinct coupling sites

-NADH is oxidized and O2 is reduced to water

-2 pump sites that diffuse H+

Q-Loop

Scalar, 2H+/2e-, E. coli UQ: cyt c oxidoreductase complex III

-Loop that scalarly pumps H+ across membrane

-Q is reduced to QH2 which is then oxidized in a cycle

-Q redox pumps/couples H+ diffusion

Q-Cycle

Vectorial, 4H+/2e-, mitochondrial bc I complex 3.

-At bc1 complex

-3 pp subunits with 2 UQ binding sites, cyt b, 2[Fe2-], 1 cyt

Q-Cycle Steps (7)

1 UQH2 oxidized when binds to bc1

2 UQ- releases e- when oxidized (e- to cyt oxidase, 2H diffuse out)

3 Fully oxidized to UQ (released e- goes to BL&SBS towards - inner membrane)

4 UQ reduced to UQ- with e-

5 Cycle resets (one full loop of the Q pools)

6 UQ generated with 2e- becomes UQ-2 (another H+ diffuses out and e- to cyt oxidase)

7 UQ-2 takes up 2H+ form inner membrane, regenerates UQH2 (resets process)

bc1 complex

Complex where Q-cycle takes place

P side -> 2UQH2 + 2 cyt c(ox) --> 2UQ + 2 cyt c(red) + 4H+(out)

N side -> UQ + 2e- + 2H+(in) --> UQH2

Total -> UQH2 + 2 cyt(ox) + 2H+(in) --> UQ + 2cyt(red) + 4H+(out)

*4H+ per 1 e- since 1 UQH2 regen (1 net UQH2 used)

"Classical" ETC

Process of ETC used in mitochondria and some B like paracoccus denitrificans

includes 4 complexes sites and 3 coupling sites

4 complexes of classical ETC

I - NADH dehydrogenase

II - Succinate dehydrogenase

III - Complex III or bc1 complex

IV - Complex IV or cyt c

Complex I

NADH dehydrogenase

Complex that uses NADH:UQ oxidoreductase to couple H+ diffusion with NADH reduction

-> 4H+/2e-

Complex II

succinate dehydrogenase

Complex that DOES NOT COUPLE

-Succinate:UQ oxidoreductase

-FAD cofactor and 3 Fe-S clusters in binding site

-Oxidizes succinate to fumarate

Complex III

bc1 complex (Q-cytochrome c oxidoreductase)

-Couples QU redox with cyt c oxidoreductase

-4H+/2e-

Complex IV

cytochrome c (aa3) oxidase

Oxides O2 to H2O

-2H+/2e-

Coupling Sites

Sites used to form electrochemical gradient vary among bacterial species and environmental condition

-Where H+ and e- transduction are linked

Total ATP per e- carrier

NADPH - 3 ATP

FAD - 2 ATP

ETC in Aerobes

Use O2 as terminal acceptor (use bd, bd-I, and bd-II oxidases depending on [O2])

ETC in Anaerobes

Use Terminal reductases (e.g., nitrate, nitrite, and DMSO, TMAO reductases)

Oxidases in AEROBES

bO3, bd-I, and bd-II

bo3 oxidase

Oxidase used in good [O2] environments. Has fastest and most efficient transfer of O2 (high Km)

bd-I and bd-II oxidase

Oxidase used in low [O2] environments. Has BEST affinity for O2 (low Km)

terminal reductase

Enzyme used in (facultative) anaerobic conditions instead of oxidase to reduce compounds other than oxygen

-> Nitrate, nitrite, DMSO, TMAO

H+/O

# H+ efflux per 2e- transferred to O2

H+/ATP

# H+ influx via ATP synthase to generate 1 ATP

(e.g., for 8 c-subunit complex = 8H+/3 ATP per turn ~ 3 H+/ATP ratio)

P/O

# ATP generated per 2e- transferred to O2

(H+/O divided by H+/ATP)

3 Coupling Sites in Mitochondria (same as classical ETC)

I -> NADH dehydrogenase (4H+/2e-)

III -> cytochrome bc1 complex (4H+/2e-)

IV -> cytochrome aa3 oxidase ( 2H+/2e-)

Respiratory chain and ATP synthase inhibitors

Retonone, HQNO (targets Q site), Antiymcin (targets Q site, cyanide (targets oxidase), oligomycin dicyclohexylcarbo-

diimide

Ionophores

Molecules that uncouple PMF

-cause ions to EQUILIBRATE across membrane

-collapses electrochemical gradient

-Inhibits oxphos (no ATP synth)

-STIMULATES Respiration in short term

EX: valinomycin, nisin, dinitrophenol

ATP Synthase inhibitors

Molecules that inhibit ETC through blocking H+ flow in F0 domain

-INCREASE electrochemical gradient

-Inhibits oxphos

-INHIBITS respiration

Microbial Fuel Cells

Harvest e- produced from e-chemical gradient with electrode. VERY EASY on seafloor w/ many electron rich reductants (e.g., Fe2+, H2S) w/ plenty of O2 as e- acceptor.

(EX Shewanella oneidensis nanowire & Rhodoferax on an

electrode)

ATP synthase Mutations

Mutations in F1 or F0 domains to enhance product formation in metabolic engineering.

(can edit so cells think they're low on ATP and produce an excess of products like NADP which can be harvested)