Oxidative phosphorlylation

• Where does the ETC take place?

  inner mitochondrial membrane
• NADH has a more negative reducing potential than FADH2, thus it will be better at giving up its electrons. O2 has a positive reducing potential and will be good at accepting electrons
• Why does FADH2 not make as much ATP?

  FADH2's reducing potential isn't negative enough to transfer its electron to complex I, so it misses out pumping protons at complex I. Proton pumping creates the ion gradient that drives ATP synthase
• What is iron's role in the ETC?

  Iron is present in the prosthetic groups of all 4 complexes and cytochrome C.
• How is iron helping transfer electrons through the complexes and electron taxis if it has a positive reducing potential?

  Redox potential depends on its environment, so when FE is attached to a prosthetic group, its redox potential can be negative. Iron in the ETC appears as:

  • Iron sulfur clusters
  • Heme groups
• What is copper's role in the ETC?

  Copper is a prosthetic group in complex IV that also helps in accepting electrons
• Which complex has both copper and iron has prosthetic groups?

  Complex IV
• What is Coenzyme Q (ubiquinone) in the ETC?

  It is an electron taxi that shuttles electrons from complex I to complex III as well as electrons from complex II to complex III.
• Why is ubiquinone able to diffuse through the membrane freely?

  It has a hydrophobic tail
• which structural part of ubiquinone is important for gaining electrons and protons?

  Quinone ring
• Which 3 complexes pump protons?

  Complexes I, III, and IV pumps H+
• Which side are the protons being pumped into of the mitochondria?

  Intermembrane space
• What electron carrier does NADH first pass its electrons to in complex I?

  Flavin mononucleotide (FMN) is the electron carrier reduced by NADH, but is subsequently oxidized by the next electron carrier in the chain until O2 is finally reduced.
• The members of the ETC are arranged so that the electrons always flow to carriers with more positive reduction potentials
• How many electrons reduce ubiquinone?

  2
• Complex I

  After NADH is oxidized and reduces FMN before FMN reduces iron-sulfur clusters, two electrons end up reducing Q and causes a structural rearrangement in the Q binding site that makes protons (from Lys and glu) more likely to bind to Q to form its fully reduced form, QH2.

  • once fully reduced, QH2 leaves complex I and dissociates into the membrane
  • since protons were taken from the lys-glu pairs by Q, the remaining protons undergo a redistribution that causes 2 H+ to be taken up from the matrix.
  • The two H+ from the matrix cause electrostatic repulsions that force 4 H+ into the IMS
• Complex II...

  FADH2 made from reaction catalyzed by succinate dehydrogenase remains on enzyme, and transfers electrons to iron-sulfur clusters until it is accepted by Q. Q accepts to H+ from the matrix to form its fully reduced form, QH2.
• Complex III...


  Electrons from QH2 from complexes I and II are passed to cytochrome C. However, QH2 has 2 electrons but cytochrome C carries only 1 electron so the electrons must pass through the Q cycle.
• Explain the first half of the Q Cycle of complex III

  The first QH2 gives up 2 electrons and 2 H+, with the H+ being released to the IMS and the 2 electrons travelling to different destinations within the complex. One of those electrons travels to Cyt C (thus reducing Cytc C), and the other electron travels through heme complexes to an oxidized Q in the second binding site until it forms a semiquinone radical. The semiquinone radical leaves and re-enters the Q pool
• Explain second half of the Q cycle...

  A second QH2 binds to a Q binding site on complex III and releases 2 electrons and 2 H+ (goes to IMS). One of its electrons is passed to cytochrome c, while the other passes through heme groups until it is accepted by the semiquinone radical from the Q pool in stage 1. After the electron is accepted by the semiquinone radical, the anion takes up two protons from the matrix to form QH2 which will join the Q pool.

  • The removal of the two H+ from the matrix and 4 H+ released into the IMS helps form the proton gradient.
• Where does Cytochrome C shuttle electrons to and from?

  Complex III to complex IV
• Complex IV...

  4 cyctochrome c's are needed to reduce oxygen to water:

  1. Two reduced cyt c are oxidized and reduce the prosthetic groups copper and iron. When the prosthetic groups are in its reduced form, it can bind to O2.
  2. As O2 binds, it forms a peroxide bridge between the two cofactors and takes an electron from each cofactor
  3. Two more molecules of cyt c. bind and 2 electrons and 2 H+ from the matrix cleave the peroxide bridge
  4. 2 more H+ from the matrix enter and cause the release of 2 molecules of H2O. Extra free energy from reduction of O2 to H2O to pumps 4 more H+ from the matrix
• How many H+ does complex IV remove?

  8
• Most of the ETC is organized into a respirasome, what is that?


  The respirasome consists of two copies of complex I, III, and IV and two copies of ATP synthase
• Proton motive force is composed of a chemical gradient and a charge gradient, explain how each can be represented in the matrix


  • The chemical gradient can be represented as a pH gradient where the matrix generally has a higher pH due to less H+.
  • The charge gradient is created by the positive charge of H+ being unequally distributed along the IMM
• Structure of ATP synthase

  A) exterior column

  B) hexamer catalysis (beta)

  C) central stalk

  
• What is the importance of the beta subunit of ATP synthase?


  It performs each of the three steps - trapping ADP and Pi, ATP synthesis, and ATP release and ADP and Pi binding by changing conformation.

  • Beta subunits can be in the L (loose) conformation that binds ADP + Pi, the T (tight) conformation that converts ADP to ATP, and the O (open) conformation that allows ATP to leave and ADP to bind
• What is the significance of the gamma subunit of ATP synthase?

  The rotation of the gamma subunit by 120 degrees converts the beta subunit into its different conformations.
• How does proton flow drive the rotation of the gamma subunit?

  • Subunit A has two hydrophobic half channels (do not span the membrane) that directly interacts with one c subunit. H+ enter the half channel but cannot move completely across the membrane.
  • The glutamic acid of subunit c is protonated by H+ as they enter the half channel, and the protonated Glu rotates through the hydrophobic space until the next subunit C arrives at the half channel and become protonated.
  • When a protonated Glu arrives at half channel, it releases its H+ to leave to the matrix though the half channel
• How is cytoplasmic NAD+ regenerated under aerobic conditions if IMM is impermeable to NAD+/NADH?

  Electrons from NADH are carried across the mitochondrial membrane and introduced to ETC by shuttles.

  • glycerol 3-phosphate shuttle (muscle): a pair of electrons from cytoplasmic NADH are transferred to glycerol 3-phosphate and then to FAD which gives it to Q. This transfer deoxidizes NAD+ and reduces Q, and the electrons enter the ETC as QH2. Yields only 1.5 ATP because FAD is used as a carrier.
  • Malate-aspartate shuttle
• • Malate-aspartate shuttle (heart/liver): electrons from NADH transferred to oxaloacetate, forming malate which is transported back into the matrix. In the matrix, malate is converted to oxaloacetate which regenerates NAD+ in the process