Cell Bio Making ATP

oxidative phosphorylation overview

- ETC transfers electrons through complexes I-IV

- proton pumping into intermembrane space

- ATP synthase uses gradient to make ATP

pathway integration of making ATP

glycolysis -> TCA -> ETC -> ATP

glycolysis

cytosolic ATP + NADH (Malate-Aspartate Shuttle)

TCA cycle (Krebs cycle) important components

NADH, FADH2, GTP

ETC purpose

oxidizes carriers, pumps protons

ATP synthase function

converts PMF into ATP

electron donors

- NADH donates at Complex I

- FADH2 donates at Complex II

- different entry points -> different ATP yields

glycolysis: ATP yield distribution from glucose in cells

2 ATP gained + 2 NADH

pyruvate oxidation: ATP yield distribution from glucose in cells

2 NADH

TCA cycle: ATP yield distribution from glucose in cells

- 6 NADH

- 2 FADH2

- 2 GTP/ATP

oxidative phosphorylation: ATP yield distribution from glucose in cells

majority of ATP

~26 ATP

theoretical yield from glucose: ATP yield distribution from glucose in cells

36

actual yield from glucose: ATP yield distribution from glucose in cells

~30-32

ETC overview: complexes I-V

- four main complexes in inner membrane

- transfer electrons from NADH/FADH2 to O2 at the end of chain

- proton pumping to IMS at Complexes I, III, IV

Peter Mitchell's Chemiosmotic Hypothesis

- proposed in 1961

- proton gradients drive ATP synthesis

- radical shift from chemical intermediates model (substrate level phosphorylation to generate ATP)

Racker and Stoeckenius experiment

- reconstituted vesicles with bacteriorhodopsin (proton pump) + ATP synthase

- light-driven proton pumping

- ATP synthesized without ETC

Complex I (NADH Dehydrogenase)

- entry point for NADH electrons

- donates 2 electrons to FMN reducing it to FMNH2

-> iron-sulfur clusters

- passes electrons down to ubiquinone (Q), reducing it to ubiquinol (QH2)

- pumps 4 protons into intermembrane space due to conformational changes as electrons move through the enzyme

Complex II (Succinate Dehydrogenase)

- dual role in TCA and ETC

- FAD accepts electrons from Succinate to generate FADH2

- ultimately transfers electrons to ubiquinone (Q) to generate ubiquinol (QH2)

-> iron-sulfur clusters/heme

- does not pump protons

ubiquinone (coenzyme Q10) shuttle

- lipid soluble electron carrier

-> 0.5-3% of the total lipid content of the IMM depending on cell type

- carries protons across/through the IMM

- shuttles 2 electrons as ubiquinol (QH2) between Complexes I/II to Complex III

Complex III: Q Cycle and Cytochrome C

- accepts electrons from QH2

- pumps 4 protons via Q cycle

- passes one electron to cytochrome c at a time

cytochrome c shuttle

- small, soluble heme protein, present in high concentrations around the IMM

- binds the intermembrane side of the IMM directly with cardiolipin, an acidic phospholipid

- transfers 1 electron at a time

- connects Complex III to IV

Complex IV (Cytochrome C Oxidase)

- Complex IV pumps 4 protons per O2 reduced

- reduces O2 -> H2O

1. accepts 2 electrons from 2 cytochrome c attachments

-> one moves to a terminal Cu, one to a Fe after Cu is full

2. reduced Fe and Cu uptake O2 and form a peroxide bridge

3. 2 additional cytochrome c attachments bring 2 more electrons

-> collects 2 H+ from matrix and forms hydroxides

4. 2 more H+ come from the matrix to produce 2 water molecules

electron transfer sequence proceeds energetically "downhill"

stepwise redox transfers release energy

{NADH-CoQ reductase} -> {Succinate-CoQ reductase} -> {CoQH2-cytochrome c reductase} -> {cytochrome c oxidase}

electron transfer sequence proceeds energetically "downhill": NADH

NADH -> Complex I -> Q -> Complex III -> cytochrome c -> Complex IV -> O2

electron transfer sequence proceeds energetically "downhill": FADH2

FADH2 enters at Complex II -> Q -> Complex III -> cytochrome c -> Complex IV -> O2

importance of oxygen as final electron acceptor

- O2 accepts all electrons from ETC and generates H2O

=> 2 O2 -> 2 H2O

- keeps ETC flowing, without it NADH accumulates

- lack of O2 in ischemia stops ATP synthesis

=> clinical oxygen deprivation causes rapid ATP deletion and cell death

cyanide inhibition of Complex IV

- cyanide binds Complex IV

- blocks electron transfer to O2

- collapses PMF -> no ATP

- rapidly fatal: seizures, coma, cardiac arrest appear in minutes due to ATP depletion in the brain and heart

Complex I: proton pumping summary

4 protons

proton pumping summary: Complex II

none

proton pumping summary: Complex III

4 protons

proton pumping summary: Complex IV

2 protons

proton motive force (PMF)

energy stored in gradient

PMF = ΔΨ + ΔpH

-> ΔΨ = electrical potential

-> ΔpH = proton concentration gradient

-> drives ATP synthase and other processes

contribution of membrane potential (ΔΨ) to PMF

- negative charge inside matrix due to charge separation of H+

- electrical force strongly pulls protons back across inner membrane to the matrix

- major contributor to PMF (~70%)

contribution of proton gradient (ΔpH) to PMF

- matrix ~0.5-1 pH unit higher than intermembrane space

- adds chemical component to PMF

- protons flow "downhill" into matrix

-> always towards equilibrium (high to low concentrations)

ΔΨ value in mitochondria

~150-200 mV

ΔpH value in mitochondria

~0.5-1 unit

- contributes ~30-60 mV

total PMF value in mitochondria

~180-220 mV

other uses of PMF in bacteria

- powers flagellar rotation through proton influx

- drives nutrient uptake and efflux pumps

- energizes transport of ions and metabolites

ΔΨ vs ΔpH dominance in mitochondria

ΔΨ dominant (~70%)

ΔΨ vs ΔpH dominance in chloroplasts

ΔpH dominant

ΔΨ vs ΔpH dominance in bacteria

balance varies with environment

ATP synthase overview (F0F1 complex)

- large enzyme spanning inner membrane

- F0 = proton channel

- F1 = catalytic head

- couples proton flow -> ATP synthesis

F0 proton channel (c-ring)

- F0 embedded in membrane

- proton binding drives c-ring rotation

- ring size (stoichiometry of subunits) affects H+/ATP ratio

F1 catalytic head (α3β3 hexamer)

- matrix-facing catalytic domain

- 3 αβ pairs form nucleotide-binding sites

- site of ATP synthesis

γ subunit

central rotating stalk

γ subunit rotation

- interacts with β-subunits of F1 catalytic head

- drives binding-change mechanism

rotary catalysis model

- proton flow rotates c-ring and the γ-subunit

- produces ATP in cyclic manner

- induces conformational changes in β-subunits of F1

binding-change mechanism

each individual β-subunit of F1 cycles through: open -> loose -> tight

loose

ADP + Pi bond

tight

ATP formed

open

ATP released

experimenal models of rotary motion

- single molecule fluorescence imaging (attach GTP)

- visible rotation of actin filament attached to γ-subunit

- confirmed rotary catalysis model

energetics of proton flow

- ~3-4 protons required per ATP

-> allows γ-subunit to interact with catalytic subunits as it rotates enough to meet the next β subunit of F1

- PMF energy converted into chemical bond energy

ATP synthase as a molecular motor

- proton flux = "fuel"

- rotation of c-ring and γ-subunit = "mechanical motion"

- ATP synthesis = "output work"

uncouplers

collapse the proton gradient

- lipid soluble molecules shuttle protons without ATP synthase

- dissipate PMF -> no ATP made

- energy lost as heat

physiological uncoupling

- brown adipose tissue and UCP1

- energy released as heat (thermogenesis)

brown adipose tissue (fat)

rich in mitochondria

UCP1

uncouples ETC from ATP synthesis (membrane protein)

DNP and obesity

- DNP used as weight loss drug in 1930s

- increased metabolism, but fatal overheating

- illustrates danger of uncoupling

P/O ratios for NADH vs FADH2

- per NADH: 10 protons pumped (~2.5 ATP total)

- per FADH2: 6 protons pumped (~1.5 ATP total)

- difference due to entry points and proton pumping various regulations of ETC

efficiency of oxidative phosphorylation

- ~30-32 ATP per glucose (theoretical yield)

- efficiency impacted by stoichiometry subunits within the c-ring

-> more rings, more H+ per ATP

- overall efficiency ~34% (rest lost as heat)

-> complete glucose oxidation: ~686 kcal/mol

-> ATP hydrolysis: ~7.3 kcal/mol per ATP

- balances energy production and thermoregulation

mitochondrial diseases

- caused by defects in mitochondrial or nuclear genes

- affect high-energy tissues (muscle, brain, heart)

- often multi-systemic and progressive

key neurological clinical features of mitochondrial diseases

- seizures

- stroke-like episodes

- developmental delay

key muscular clinical features of mitochondrial diseases

- weakness

- exercise intolerance

misc. key clinical features of mitochondrial diseases

- hearing loss

- cardiomyopathy

- endocrine dysfunction

classic mitochondrial diseases

- MELAS

- LHON

- MERRF

MELAS

mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes

LHON

Leber's hereditary optic neuropathy

MERRF

myoclonic epilepsy with ragged red fibers

mechanisms behind pathology of mitochondrial diseases

- reduced ATP production -> energy crisis

- increased ROS generation -> oxidative damage

- defective apoptosis regulation through signaling can lead to neurodegeneration

diagnosis of mitochondrial diseases

- muscle biopsy

- genetic testing

- biochemical assays

supportive treatments of mitochondrial diseases

- exercise

- diet

- cofactor supplements

experimental therapies for mitochondrial diseases

- gene therapy

- mitochondrial replacement