Chapt. 21 oxidative phosphorylation

Chapt. 21 oxidative phosphorylation

Ch. 21 oxidative phosphorylationStudent Learning Outcomes:

• Explain process of generation of ATP by oxidative phosphorylation:

• NADH + FAD(2H) donate e- to O2 -> H2O• ATP synthase makes ATP (~3/NADH, ~2/FAD(2H)

• Describe chemiosmotic model, H+ gradient• Describe complications of deficiency of ETC –

anemia, cyanide, OXPHOS diseases• Describe transport through mitochondrial

membranes

I. Oxidative phosphorylation summary

Oxidative phosphorylation overview:• Multisubunit complexes I, coQ, III, IV pass e- to O2

• H+ are pumped out -> electrochemical gradient• H+ back in through ATP synthase makes ATP

Fig. 1

Proton Motive Force

Proton motive force: • Electrochemical potential gradient• Membrane is impermeable to H+• pH gradient ~ 0.75 pH units

Fig. 2

ATP synthase

Figs. 3,4ATP synthase (F0F1 ATPase):• F0 inner membrane (12 C)• F1 matrix has stalk, headpiece• H+ go through a-c channel• 12 protons/turn -> 3 ATP• Binding change mechanism:

• Turning releases ATP

B. Components of Electron Transport Chain

Components of Electron Transport chain:• Series of transfers of e- down energy gradient

• Series of oxidation reduction reactions• e- finally to O2 -> H2O

• H+ pushed across membrane

Fig. 5

Components of Electron Transport Chain

NADH dehydrogenase: 42 subunits, • FMN binding proteins• Fe-S binding proteins (transfer single e-)• binding site for CoQ • pass e- to CoQ; transfers 4 H+

Fig. 5,6

Components of Electron Transport Chain

Complex II: succinate dehydrogenase (from TCA)• FAD bound e- from TCA, • Other FAD from other paths • Not sufficient energy to transfer H+ when pass e- to CoQ

Fig. 5

Coenzyme Q

Coenzyme Q is not protein bound. 50-C chain inserts in membrane, diffuses in lipid layer

• Also called ubiquionone (ubiquitous in species)• Transfer of single e- makes it site for generation of toxic

oxygen free radicals in body

Fig. 7

Cytochromes have heme groups

Cytochromes have heme groups:• Proteins with hemes• Fe3+ -> Fe2+ as gain e-• Transfer e- to lower potential

Figs. 5,8; Heme A is in Cyt a, Cyt a3

C. Pumping of protons not well understood

Cytochrome C oxidase: Cyt a, Cyt a3, O2 binding:• Receives e- from Cyt c (takes 4 to make 2 H2O)• Transfers to O2; Pumping of H+ not well understood; must couple to

e- transport and ATP; otherwise backup

Fig. 5

D. Energy yield

Energy yield from oxidation by O2:NADH: Dg0’ ~ -53 kcal; FAD(2H) ~ -41 kcalEach NADH 2e- -> ~ 10 H+ pumped; Takes ~ 4 H+/ATP -> 2.5 ATP/ NADH; 1.5/ FAD(2H) or if ~ 3H+/ATP -> 3 ATP/ NADH; 2/ FAD(2H)

Fig. 5

E. Inhibition of chain, sequential transfer

Once start ETC, must complete transfer of e-• In absence of O2, backup since carriers full of e-• Inhibitors like cyanide (binds Cyt c oxidase) mimics anoxia: prevents proton pumping• Cyanide binds Fe3+ in heme of Cyt a a3

• CN in soil, air, foods (almonds, apricots)

Fig. 5

OXPHOS diseases from mutated mitochondrial DNAHuman mt DNA is 16.569 kb:13 subunits of ETC: 7 of 42 of Complex I 1 of 11 Complex III 2 of ATPsynthase22 tRNA, 2 rRNA

OXPHOS diseases from mutated Mitochondrial DNA

Table 21.1 examples OXPHOS diseases from mt DNA

Point mutations in tRNA or ribosomal RNA genes:

MERFF (myoclonic epilepsy and ragged red fiber):• tRNAlys progressive myoclonic epilepsy, mitochondrial myopathy

with raged red fibers, slowly progressive dementia

• Severity of disease correlated with proportion mutant mtDNA

LHON (Leber’s hereditary optic neuropathy):• 90% of cases from mutation in NADH dehydrogenase

• Late onset, acute optic atrophy

Nuclear genes can cause OXPHOS

Mutated nuclear genes can cause OXPHOS:• About 1000 proteins needed for Oxidation

phosphorylation are encoded by nuclear DNA.• Electron transport chain, translocators

• Need coordinate regulation of expression of genes, import of proteins into mitochondria, regulation of mitochondrial fission

• Nuclear regulatory factors

for transcription in nucleus, mt• Often recessive autosomal

III. Coupling of electron transport and ATP synthesis

Fig. 10

Concentration of ADP controls O2 consumption:• Or phosphate potential ( [ATP]/[ADP][Pi])

1.ADP used to form ATP2.Release ATP requires H+ flow3.H+ decreases proton gradient4.ETC pumps more H+, uses O2

5.NADH donates e-, makes NAD+ to return to TCA cycle or other

Uncoupling agents dissipate H+ gradient without ATP

Uncoupling agents decrease H+ gradient without generating ATP:

Ex. DNP is a chemical uncoupler:• lipid soluble, carries H+ across membrane

Fig. 11

Uncoupling proteins form channels, thermogenesis

Uncoupling proteins form channels for protons:• Ex. UCP1 (thermogenin) makes heat in brown adipose

tissue (nonshivering thermogenesis); many mitochondria;• Infants have lots of brown adipose tissue, not adults

Fig. 12

IV. Transport through mitochondrial membranesTransport across inner mitochondrial membranes uses channels,

translocases:• Form of active transport using proton gradient :• ANT exchanges ATP: ADP • Symport H+ with Pi• Symport H+, pyruvate

Fig. 13

Transport across outer membrane:

Fig. 13

Transport across outer membrane:Rather nonspecific pores:• VDAC voltage-dependent anion channels• Often kinases on cytosolic side

Mitochondrial permeability transition pore

Fig. 14

Mitochondrial permeability transition pore:• Large nonspecific pore: • Will lead to apoptosis (cell death)• Highly regulated process

• Hypoxia can trigger

• Pore opens, lets H+ flood in,• Anions, cations enter• Mitochondria swell and • Irreversible damage

Key concepts

• Reduced cofactors NADH, FAD(2H) donate e- to electron transport chain

• ETC transfers e- to O2 -> H2O• As e- transferred, H+ pushed across membrane;• H+ gradient used by ATP synthase to make ATP

• O2 consumption tightly coupled to ATP synthesis• Uncouplers disrupt process – poisons

• OXPHOS diseases from mutations in mt DNA or in nuclear DNA

• Compounds transported across mt membranes

Review question

5. Which of the following would be expected for a patient with an OXPHOS disease?

A.A high ATP:ADP ratio in the mitochondria

B.A high NADH:NAD+ ratio in the mitochondria

C.A deletion on the X chromosome

D.A high activity of complex II of the electron-transport chain

E. A defect in the integrity of the inner mitochondrial membrane

Review question p. 392

Decreased activity of the electron transport chain can result from inhibitors as well as from mutations in DAN. Why does impairment of the ETC result in lactic acidosis?

• Inhibit ETC -> Impaired oxidation of pyruvate, fatty acids and other fuels; therefore more lactate and pyruvate in blood.

• NADH oxidation requires complete transfer of e- to O2, so defect in chain increase NADH:NAD+ and inhibit pyruvate dehydrogenase.