Chemiosmosis and G3P shuttle



The generation of ATP by the movement of H+ across a membrane during cellular respiration. ATP synthase allows protons to pass through the membrane and uses the kinetic energy to phosphorylate ADP, making ATP.

  • The membrane must be impermeable to ions so that a proton concentration gradient can be maintained.
  • Specific transporters allow movement of ions across the membrane.
  • Electron transport through the ETC creates a proton concentration gradient in which the cystolic side of the inner mitochondria membrane has a higher conc of protons.
  • ATP synthase catalyzes ADP phosphorylation driven by the movement of protons across the inner membrane from the cytosol to the matrix.
  •  NAD and FAD are carriers pass electrons to the ETC in the inner mitochondrial membrane, which in turn pass them to other proteins in the ETC. The energy available in the electrons is used to pump protons from the matrix across the inner mitochondrial membrane, storing energy in the form of a transmembrane electrochemical gradient.
  • The protons move back across the inner membrane through the enzyme ATP synthase.
  • The flow of protons back into the matrix of the mitochondrion via ATP synthase provides enough energy for ADP to combine with inorganic phosphate to form ATP.
  • The electrons and protons at the last pump in the ETC are taken up by oxygen to form water.


Evidence to support postulates: 

Postulates explain the effect of uncouplers which bind to the H+ and carries them across the membrane to the matrix, thereby affecting the proton conc gradient. The second postulate demonstrates that the medium becomes acidic when mitochondria under anaerobic conditions are given small amounts of oxygen. Acidifying the media can form ATP from ADP and Pi without the need for electron transfer. ATPase activity in damaged mitochondria acts in reverse using the proton gradient to synthesize ATP i.e an ATP synthase.

G3P Shuttle:

This is an alternative mechanism that leads to the reduction of Ubiquinone.

Glycerol-3-Phosphate is converted to Dihydroxyacetone phosphate (irreversible). While this is done, mitochondrial G3P Dehydrogenase reduces E-FAD to E-FADH2 which when oxidized back to E-FAD causes Q to be reduced to QH2 which enters into oxidative phosphorylation.  When Dihyoxyacetone is reconverted to G3P (reversible), NADH + H+ is used and oxidised to NAD+.


Q Cycle and Chemiosmosis


Q Cycle:

The modified Q cycle in Complex III results in the reduction of Cytochrome c, oxidation of ubiquinol to ubiquinone, and the transfer of four protons into the intermembrane space, per two-cycle process.

Chemical Reactions:

The first reaction of Q cycle is:
CoQH2 + cytochrome c1 (Fe3+) → CoQ- • + cytochrome c1 (Fe2+) + 2 H+ (intermembrane)

Then the second reaction of the cycle involves the reduction of the transient semiquinone by another electron to give CoQH2:
CoQH2 + CoQ- • + cytochrome c1 (Fe3+) + 2 H+ (matrix) → CoQ + CoQH2 + cytochrome c1 (Fe2+) + 2 H+ (intermembrane)

Combining the two equations, we have the overall reaction of Q cycle:
CoQH2 + 2 cytochrome c1 (Fe3+) + 2 H+ (matrix) → CoQ + 2 cytochrome c1 (Fe2+) + 4 H+ (intermembrane)

Explanation of Reactions: 

Ubiquinol (QH2) binds to the Qo site of complex III via hydrogen bonding to His182 of the Rieske iron-sulfur protein and Glu272 of Cytochrome b. Ubiquinone (Q), in turn, binds the Qi site of complex III. Ubiquinol is divergently oxidized (gives up one electron each) to the Rieske iron-sulfur ‘(FeS) protein’ and to the bL heme. This oxidation reaction produces a transient semiquinone before complete oxidation to ubiquinone, which then leaves the Qo site of complex III.
Having acquired one electron from ubiquinol, the ‘FeS protein’ is freed from its electron donor and is able to migrate to the Cytochrome c1 subunit. ‘FeS protein’ then donates its electron to Cytochrome c1, reducing its bound heme group.[1][2] The electron is from there transferred to an oxidized molecule of Cytochrome c externally bound to complex III, which then dissociates from the complex. In addition, the reoxidation of the ‘FeS protein’ releases the proton bound to His181 into the intermembrane space.
The other electron, which was transferred to the bL heme, is used to reduce the bH heme, which in turn transfers the electron to the ubiquinone bound at the Qi site. The movement of this electron is energetically unfavourable, as the electron is moving towards the negatively charged side of the membrane. This is offset by a favourable change in EM from -100 mV in BL to +50mV in the BH heme. The attached ubiquinone is thus reduced to a semiquinone radical. The proton taken up by Glu272 is subsequently transferred to a hydrogen-bonded water chain as Glu272 rotates 170° to hydrogen bond a water molecule, in turn hydrogen-bonded to a propionate of the bL heme.
Because the last step leaves an unstable semiquinone at the Qi site, the reaction is not yet fully completed. A second Q cycle is necessary, with the second electron transfer from cytochrome bH reducing the semiquinone to ubiquinol. The ultimate products of the Q cycle are four protons entering the intermembrane space, two protons taken up from the matrix and the reduction of two molecules of cytochrome c. The reduced cytochrome c is eventually reoxidized by complex IV. The process is cyclic as the ubiquinone created at the Qi site can be reused by binding to the Qo site of complex III.

Electron Transport Chain

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.

Complex I:

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.

Complex II:

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.

Complex III:

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.

Redox Chemistry:

  • 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.

Complex IV:

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