Oxidative Phosphorylation I & II Flashcards
Describe the ultrastructure of a mitochondria.
• Have 2 membrane systems
– Outer membrane (permeable due to presence of porin aka VDAC)
– Inner membrane – (impermeable, has metabolite
transporters, folded into series of cristae)
• Two compartments
– Intermembrane space
– Matrix (site of TCA cycle and fatty acid oxidation)
• Oxidative phosphorylation – inner membrane
Describe the mitochondria genome.
• Mitochondria semi autonomous organelles
• Endosymbiotic relationship with host cell
• Free living organism engulfed by another cell
• Genome ranges in size between species
• Sequence data shows all mito. derived from R.
prowazekii due to a single endosymbiotic event
• Have their own DNA, make proteins and RNAs
• Human mito DNA has 16, 569 bp and encodes
13 respiratory chain proteins, rRNAs, tRNAs
What is the overview of oxidative phosphorylation?
- TCA cycle generates NADH and FADH2
- In oxphos these high energy electrons flow through 4 protein complexes called the electron transfer chain (ETC)
- Electrons reduce molecular O2 to water
- Three of the complexes pump protons from matrix to intermembrane space
- Protons return to matrix by flowing through another complex called ATP synthase powering the synthesis of ATP
What are the components of the electron transport chain?
• Electrons transferred from NADH to O2 via three large protein complexes
– NADH Q oxido reductase
– Q cytochrome c oxidoreductase
– Cytochrome c oxidase
• Electron flow exergonic
• Powers flow of protons across inner membrane
• Fourth complex is succinate Q reductase
• Has succinate dehydrogenase which FADH2 generates in TCA cycle
• Electrons from FADH2 enters through Q cytochrome c oxidoreductase
• Succinate Q reductase does not pump protons
List and describe the electron carriers.
- Coenzyme Q aka ubiquinone
* Transfers electrons from NADH Q oxidoreductase and the Succinate Q reductase to Q cytochrome c oxidoreductase
* Coenzyme Q has long tail made of 5 – C isoprene units which makes it hydrophobic
* Most common is CoQ 10 - Cytochrome c
* Shuttles electrons from Q cytochrome c oxidoreductase to cytochrome c oxidase
* Final component of the chain
* Catalyzes reduction of O2
List iron sulfur clusters?
- Single iron ion tetrahedrally coordinated to SH groups of four Cys residues of the protein
- Two iron ions, two inorganic sulfides and four Cys residues (2Fe-2S)
- Four iron ions, two inorganic sulfides and four Cys residues (4Fe-4S)
What is Friedreich’s Ataxia?
- Mutations in the protein Frataxin
- Loss of function of
- Small mitochondrial protein crucial for the synthesis of Fe-S clusters
- Affects the CNS and PNS as well as heart and skeletal system
- Most common mutation is trinucleotide expansion in Frataxin gene
Describe complex I
- First point of entry of electrons from NADH
- Complex I aka NADH dehydrogenase aka NADH-Q oxidoreductase
- Large protein (>900 kDa, with 46 polypeptide chains)
- Encoded by nuclear and mitochondrial genes
- L shaped with a horizontal arm lying in the inner membrane and vertical arm that projects into matrix
Describe what happens in complex II
- FADH2 enters the ETC through Succinate-Q reductase complex (Complex II)
- FADH2 does not leave the complex
- Its electrons transferred to FeS and then to Q to form QH2
- Does not pump protons
- Consequently less ATP synthesized from oxidation of FADH2
Describe what happens in complex III
- Electrons from QH2 are passed on to cytochrome c by cytochrome c reductase aka Complex III
- Flow of electrons through this complex leads to transport of 2 protons to cytoplasmic side
Describe Cytochrome C Oxidase
• Last complex aka Complex IV
• Cyt c oxidase catalyzes transfer of electrons
from reduced Cyt c to molecular oxygen, the final acceptor
• Makes these reactions aerobic
• Makes humans “breathe”
• Four electrons funneled to oxygen to reduce it to water
• Concomitantly protons are pumped from matrix to cytoplasmic side of inner membrane
Describe free radicals
- Complete reduction of oxygen forms water
- Partial reduction forms dangerous species
- Transfer of a single electron to oxygen forms superoxide anion
- Transfer of 2 electrons to oxygen forms hydrogen peroxide
- Hydroxyl radical formed from both
Name two antioxidants
Superoxide dismutase (SOD)
Catalase
Describe the Chemiosmotic Hypothesis
- ETC accompanied by transport of protons from matrix to cytoplasmic side of inner membrane
- Generates a pH gradient and membrane potential
- Constitutes a proton motive force
- Used to drive ATP synthesis
Give the Evidence for the Chemiosmotic Hypothesis
- Synthetic phospholipid vesicles made
- Purified ATP synthase and bacteriorhodopsin reconstituted into the vesicles
- Incubated with ADP + Pi
- Exposed to light
- ATP generated
Describe the structure of ATP Synthase
• Aka Complex V
• Embedded in inner membrane
• Ball and stick structure
• F0 subunit is stick – embedded in membrane
• Has a proton channel
• F1 subunit is ball, protrudes into matrix side
• Contains catalytic domains
• F1 subunit made of 5 types of polypeptide chains with
different stoichiometries
• Alpha3, Beta3, gamma, delta and epsilon
• Alpha and beta arranged alternately in a hexameric ring
• Both bind nucleotides but only the beta are catalytically active
• Above the alpha and beta is a stalk made of gamma and epsilon proteins
• The gamma subunit has a long helical coil that extends into the center of the alpha3 and beta3 hexamer
• The gamma subunit breaks the symmetry of the 3 beta
subunits making each one distinct
• F0 has a proton channel made of 8-14 c
subunits embedded in membrane
• F0 and F1 connected in 2 ways:
– 1. central gamma and epsilon stalk
– 2. Exterior column – 1 a su, 2 b su, and delta su
Explain what ATP synthase does
• ATP synthase molecules associate with each
other to form dimers
• Dimers come together to form oligomers
• Stabilizes the individual molecules to rotational forces required for catalysis
• Maintains curvature in inner membrane
• Cristae allow the proton gradient to be in close proximity to the ATP synthase
What is the Role of a proton gradient?
• ATP synthase when incubated with ADP and Pi
formed ATP in absence of a proton gradient
• Release the ATP from the synthase
What happens at the ATP synthase nucleotide binding sites?
- The 3 beta su comprise active site of ATP synthase
- The gamma su passes through center, creating asymmetry
- The proton motive force causes the 3 su to sequentially change conformation
- Conformational change alters function
- Rotation of the Gamma su switches these forms
What are the three steps in ATP synthesis?
– 1. Binding of ADP and Pi (L conformation)
– 2. ATP synthesis (T conformation)
– 3. Release of ATP (O conformation)
What are Progressive alterations of the forms of the 3 active sites?
SU 1 – L T O L T O
SU 2 – O L T O L T
SU 3 – T O L T O L
Describe the Proton conducting unit of ATP synthase
- The c subunit made of 2 alpha helices that span membrane
- An aspartic acid residue lies in the center of the membrane
- The a subunit has 2 half channels
- Allows proton to enter and pass partway but not completely
Describe the Mechanism of action of ATP-ADP translocase
- ATP and ADP not permeable across mito membrane
- Need a carrier
- ATP-ADP translocase
- Flow of ATP and ADP coupled, i.e., ADP enters matrix only if ATP leaves
Describe the Regulation of Cellular Respiration
• Levels of ATP regulate respiration
• Electrons flow through ETC only when ADP
phosphorylated to ATP
• Regulation by ADP levels called respiratory
control
How is ATP synthase regulated?
• Inhibitory factor I – inhibits hydrolytic activity of ATP synthase
• Prevents the reverse reaction, i.e., ATP breakdown
• In ischemia or oxygen deprivation
• In cancers – facilitates the switch from aerobic
to anaerobic respiration (Warburg effect)
Describe Uncoupling and Heat Generation
• Some organisms can uncouple oxphos from ATP synthesis
• Used to generate heat and maintain body temperature
(hibernating animals)
• Happens in brown adipose tissue
• Rich in mitochondria
• Reddish brown due to cytochromes and hemoglobin
• Inner mitochondrial membrane contains uncoupling protein
(UCP 1) aka thermogenin.
• Transfers protons from cytoplasm to matrix side
• Energy converted to heat instead of ATP
• UCP 2 and UCP-3 also uncouple oxphos from ATP synthesis
• Play role in energy homeostasis
How is Ox Phos inhibited?
• Inhibition of ETC
• Inhibition of ATP synthase – oligomycin
(antibiotic and antifungal agent) inhibits influx
of proteins into ATP synthase by binding to c
su
• Uncoupling electron transport from ATP
synthesis – by 2, 4 – dinitrophenol. Dissipates
the proton gradient
• Inhibition of ATP export – atractyloside and
bongkrekic acid inhibit ATP-ADP translocase
List the inhibitors of the electron transport chain.
rotenone and amytal blocks NADH-Q to QH2
antimycin A blocks Q-cytochrome c to cytochrome c
CN-, N3-, CO blocks cytochrome c to O2
