ETC couples electron transfer between an electron donor (such as NADH) and an electron acceptor (such as O2) with the transfer of H+ ions across a membrane. Protons are pumped from the mitochondrial matrix into the intermembrane space creating an electrochemical proton gradient which allows ATP synthase (ATP-ase) to use the flow of H+ through the enzyme back into the matrix to generate ATP from ADP and inorganic phosphate. The mitochondria consists of a double membrane, the outer is permeable to most small molecules and ions while the inner is impermeable to most small molecules hence specific transporters are required. ETC takes place in the inner membrane through 4 complexes. There are 5 types of electron-carrying molecules that shuttles electrons along these complexes; NAD+, FAD/FMN, Qbiquinone, Cytochromes and Iron-Sulphur Proteins.
Summary through ETC: Complex I accepts electrons from the Krebs cycle NADH, and passes them to coenzyme Q, which also receives electrons from complex II, UQ passes electrons to complex III, which passes them to cytochrome c. Cyt c passes electrons to Complex, which uses the electrons and hydrogen ions to reduce molecular oxygen to water.
In Complex I (NADH dehydrogenase) two electrons are removed from NADH to FMN to form FMNH2. Electrons are then passed one at a time (through a series of Fe-S clusters) to lipid-soluble carrier, ubiquinone (Q) to form semiquinone intermediate then QH2. The reduced product, ubiquinol (QH2) freely diffuses within the membrane, and Complex I translocates four protons (H+) across the membrane, thus producing a proton gradient.
The pathway of electrons occurs as follows:
NADH + Q + 5H+ (matrix) –> NAD+ + QH2 + 4H+
- NADH is oxidized to NAD+, by reducing Flavin mononucleotide to FMNH2 in one two-electron step.
- FMNH2 is then oxidized in two one-electron steps, through a semiquinone intermediate.
- Each electron thus transfers from the FMNH2 to an Fe-S cluster, from the Fe-S cluster to ubiquinone (Q).
- Transfer of the first electron results in the free-radical (semiquinone) form of Q, and transfer of the second electron reduces the semiquinone form to the ubiquinol form, QH2.
- During this process, four protons are translocated from the mitochondrial matrix to the intermembrane space.
In Complex II (succinate dehydrogenase) additional electrons are delivered one at a time through three Fe-S centres to Q to form QH2. Complex II consists of five prosthetic groups and four protein subunits: SDHA, SDHB, SDHC, and SDHD. C and D are integral membrane proteins with three transmembrane helices. Other electron donors (e.g., fatty acids an glycerol 3-phosphate) also direct electrons into Q (via FAD). Complex II is NOT a proton pump. b Heme does NOT play a direct role in electron transfer.
Structure: The enzyme is a homodimer with 11 distinct polypeptide chains. Major prosthetic groups; 3 hemes and a Riske 2Fe-2S cluster which mediate electron-transfer between Q in the membrane and Cytochrome C in the intermembrane space.
- In Complex III (cytochrome bc1 complex), the Q-cycle contributes to the proton gradient by an asymmetric absorption/release of protons.
- Two electrons are removed from QH2 at the QO site and sequentially transferred to two molecules of cytochrome c.
- The two other electrons sequentially pass across the protein to the Qi site where Q is reduced to QH2.
- A proton gradient is formed by two quinol (4H+4e-) oxidations at the Qo site to form one quinol (2H+2e-) at the Qi site.
- In total six protons are translocated: two protons reduce quinone to quinol and four protons are released from two ubiquinol molecules.
Structure: Dimer formed by a protein monomer composed of 13protein subunits. Three subunits form the central core of the enzyme. Major prosthetic groups includes CuA/CuA, Heme A and Heme a3-CuB. Heme a3-CuB is the site of reduction of O2 to H2O.
Redox Chemistry: In Complex IV (cytochrome c oxidase), sometimes called cytochrome A3, four electrons are removed from four molecules of cytochrome c and transferred to molecular oxygen (O2), producing two molecules of water. At the same time, four protons are removed from the mitochondrial matrix (although only two are translocated across the membrane), contributing to the proton gradient. The activity of cytochrome c oxidase is inhibited by cyanide.
Cytochrome C Oxidase Mechanism:
- Two molecules of Cytochrome C used to sequentially transfer electrons to reduce CuB and Hemea3
- Reduced CuB and Fe in Hemea3 bind O2, which forms a peroxide bridge.
- Addition of two more electrons and two more protons cleave the peroxide bridge.
- Addition of two more protons lead to the release of water.
Peroxide bridge: Oxygen bound to hemea3 is reduced to peroxide by the presence of CuB.
Total protons moved per 2e:
4H+ pumped at complex I. 2H+ taken from matrix, 4H+ released at the IMS at complex III. 2H+ pumped and 4H+ taken up from the matrix at complex IV. For NADH 10 protons pumped and for FADH2 6 protons pumped.
- Complex I: Rotenone and Piericidin A
- Complex II: Malonate
- Complex III: Antimycin A (Qn) , Myxothiazol (Qp) and Stigmatellin (RFe-S)
- Complex IV: Cyanide, CO