Oxidative Phosphorylation Flashcards
Mitochondrial membrane structure
- outer membrane (very impermeable)
- intermembrane space
- inner membrane
- matrix
What is the structure of the mitochondrial genome?
Circular (no chromatin, histones, etc)
What are the contents of the mitochondrial genome?
~17kb = 37 genes (13 proteins, 22 tRNAs, 2 rRNAs)
Describe the structure of a voltage dependent anion channel.
B barrel with a-helix that opens/closes the channel in a voltage dependent manner
Where are voltage dependent anion channels located and what do they transport?
- Outer mitochondrial membrane
- ATP/ADP, Pi, pyruvate, citrate
What are some inner membrane transporters and what are their substrates?
- Adenine Nucleotide Transporter = ATP/ADP
- Dicarboxylate carrier = malate/phosphate
- Tricarboxylate carrier = citrate + H+/malate
- Pyruvate carrier = OH-/pyruvate
- Phosphate carrier = OH-/phosphate
- Ornithine transporter = ornithine/citrulline
What key reactants/products are transported in/out of the mitochondria for oxidative phosphorylation?
IN: O2, ADP, food-derived pyruvate and FAs
OUT: CO2, ATP
What is the purpose of the glycerol-3-phosphate shuttle?
Regenerate oxidized NAD+ for glycolysis
What reaction is catalyzed by cytosolic glycerol-3-P DH?
- Reduces DHAP to glycerol-3-P, requiring 1 NADH
- Regenerates oxidized NAD+ for glycolysis
What reaction is catalyzed by mitochondrial glycerol-3-P DH?
- Glycerol-3-P is oxidized back to DHAP, generating 1 FADH
- Protein is inner-membrane bound: DHAP remains in cytosol and electrons from FADH are transferred to the ETC
What are the major and minor pathways for transporting re-oxidizing the NADH generated in glycolysis to NAD+?
Major: G3P shuttle
Minor: malate-aspartate shuttle
What is the purpose of the malate-aspartate shuttle?
Regenerate oxidized NAD+ for glycolysis
What are the steps of the malate-aspartate shuttle?
Overall: transport + carbon skeleton recycling
- cytosolic OAA reduced to malate, requiring 1 NADH (and regenerating an NAD+ for glycolysis)
- Malate transport across inner mitochondrial membrane
- Malate oxidized to OAA, generating 1 NADH (goes into ETC)
- OAA is transaminated to aspartate, requiring glu and generating a-KG
- Aspartate is transported across the inner mitochondrial membrane to cytosol
- Aspartate transaminated to OAA, requiring a-KG and generating glu
*glu and a-KG transported across inner membrane
What is the function of complex 1?
NADH dehydrogenase:
- accepts 2 electrons from NADH
- electron transfer via FMN and Fe-S clusters to CoQ
- 4 protons pumped into IM space
What is the function of complex 2?
Succinate dehydrogenase (TCA enzyme) + ETF CoQ oxidoreductase + G3PDH:
- accepts electrons from succinate
- electron transfer via FAD to CoQ
- does not span membrane nor pump protons
What is the function of complex 3?
Cytb-c1 complex:
- accepts electrons from reduced CoQ (CoQH2)
- electron transfer via cyt b and Fe-S clusters to cyt c
- cytochromes have a bound heme
- pumps 4 protons across membrane
What is the function of complex 4?
Cyt c oxidase:
- accepts electrons from cyt c
- electron transfer via Cu and Fe ions and cyt a’s to O2
- pumps 2 protons across membrane
What is ubiquinone?
- Electron carrier that moves through the membrane to transport 2 protons and 2 electrons from complexes 1 and 2 to complex 3 in its reduced form
- hydrophobic hydrocarbon tail is produced via cholesterol biosynthesis
What is cytochrome c?
- Electron carrier that transports electrons from complex 3 to complex 4
- Uses iron-sulfur clusters to complex electrons
What is the purpose of iron-sulfur clusters?
- Coordinate electrons for transport
- Heme holds iron
- Proteins fold to expose free sulfhydrils on cysteines
How does redox potential change across the ETC?
- Redox potential (electron affinity) increases
How does the energy per electron change across the ETC?
- Decreases slightly as some energy is lost as heat and also used at each step
Why are protons pumped across the inner membrane?
To create an electrochemical gradient: intermembrane space is + charged and acidic
Which direction are protons pumped?
matrix -> intermembrane space
What is the relationship between the cytoplasm and the intermembrane space?
Considered continuous because voltage gated channels allow for diffusion
What is the chemiosmotic theory of oxidative phosphorylation?
- A proton motive force is generated from the electrochemical proton gradient established across the inner membrane
- Protons want to move from the positively charged, acidic inner membrane space down the gradient into the matrix
What is the purpose of F0F1 ATP synthase?
- Transfers chemical energy (protons) into mechanical energy (rotor) back to chemical energy (ATP)
- As protons move down the gradient through the rotor, conformational changes to the protein allow for generation of ATP
Why is ATP synthase reversible?
- Can hydrolyze ATP to pump protons into the inter membrane space
- Allows electrochemical gradient/membrane potential to be maintained for other processes (other than ATP synthesis) while the cell is in a high energy state and no ATP synthesis is needed (ie ETC is inactive)
How is the rate of oxygen consumption by complex 4 controlled?
ADP concentration
What is oligomycin?
ETC inhibitor that blocks the F0 channel of ATP synthase?
What are some inhibitors of of CoQ reduction?
via complex 1: piericidin A, amobarbital, rotenone
via complex 2: carboxin, TTFA
What are some inhibitors of complexes 2, 3, and 4?
2: malonate
3: BAL, antimycin A
4: H2S, CO, CN-
How do ETC uncouplers work?
- Facilitate transport of protons across inner membrane, dissipating the gradient
- Heat is generated instead of ATP
thermogenin
- ETC uncoupling protein
- found in inner mitochondrial membrane in brown fat in infants
- transports protons back into the matrix after they are transported into the inner membrane space via the etc to produce heat
2,4-DNP
- chemical etc uncoupler
- small hydrophobic molecule binds proton in the acidic IM space and diffuses across the membrane to the matrix
- used as a weight loss drug as FAs could be burned w/o ATP generation
What are other functions of the electrochemical gradient?
- Voltage gradient: drives transport of ADP (-3) into the matrix and ATP (-4) into the IM space/cytosol
- pH gradient: phosphate (-) and pyruvate (-) import
What does it mean that the mitochondria is a calcium sink?
Takes in and releases Ca2+ ions to maintain cytosolic [Ca2+]
What is the function of the mitochondrial permeability transition pore?
- Allows free passage of water and solutes into the mitochondria => lysis
- Involved in apoptosis
What are the components of the mitochondrial permeability transition pore?
- ANT in inner membrane + VDAC in outer membrane
- CK in IM space to accept ATP
- HK in cytosol to accept ATP
What regulates mitochondrial permeability transition pore formation?
+ : atractyloside, Ca2+, Bax, PO4, ROS
- : bongkrekic acid, ATP
What reactions generate ROS?
- Fenton reaction: H2O2 + iron => hydroxyl ion + hydroxyl radical
- O2 + electron => superoxide (ROS)
- superoxide + electron + 2 protons => H2O2 which can combine with a proton and an electron to make water and a hydroxyl radical
Why is it dangerous to have free iron in the cell?
- Can generate hydroxyl radicals by reducing H2O2
- This is why we need heme, ferritin
What are some NOS’s?
- nitric oxide = free radical
- peroxynitrite = strong oxidizing agent
- peroxynitrous acid
- nitronium ion = nitrating agent
- nitrogen dioxide = free radical
- nitrogen trioxide = nitrosating agent
What are some sources of NOS’s?
- Nitric oxide synthase generates nitric oxide with conversion of arginine to citrulline
- Diet and gut bacteria generate nitrite which can be converted to nitrogen trioxide
What are some non-enzymatic antioxidants?
- vitamins C and E
- carotenoids (dietary)
- flavonoids (dietary)
- uric acid (purine catabolism)
- melatonin
What antioxidant enzymes defend against oxygen radicals?
- superoxide dismutase (cytosol and mitochondria) converts 2 superoxide into O2 + H2O2
- catalase (peroxisome) converts 2 H2O2 into 2 H2O + O2
- glutathione peroxidase reduces H2O2 to H2O with the oxidation of 2 GSH
What is glutathione?
- GSH = gly + cys + glu
- the free sulfhydryls on the cys residues of 2 GSH will form a disulfide bond that can be used as a reducing agent
How is reduced 2 GSH regenerated from GSSG?
Glutathione reductase reduces GSSG using NADPH
What cellular damage is caused by ROS?
- Protein damage => aggregation
- DNA damage => apoptosis, cancer
- Membrane lipid peroxidation => “contagious” as damaged lipids act as free radicals too => change in membrane permeability
- Membrane damage => Ca2+, Na+, and water influx => cell swelling
Generally, how are ROS and NOS generated?
oxidative metabolism
