Electron Transport System & Oxidative Phosphorylation

The details of the molecular complexes involved in this set of reactions have recently been illuminated in great detail. However, the mechanisms of the hydrogen ion 'pumps' associated with complexes I, II and IV are still being investigated. Keep in mind that, at this point, the literature still contains many versions of how this system works and the number of ATP molecules resulting per breakdown of one glucose molecule. This summary will only consider the system as presented here.

The final phase occurs within the mitochondria. The cut-away illustration of a mitochondrion shows a large inner membrane with folds called cristae. The center is a fluid-filled matrix that contains the soluble enzymes of Krebs' cycle. There is a narrow space between the inner and outer membranes (not labeled) called the intermembrane space (IMS).

The schematic illustrations shows five large protein complexes that are embedded within the inner mitochondrial membrane. Current research suggests several ways they might be grouped but there is no consensus. Many copies of each complex likely diffuse slowly within the membrane. Also within the inner membrane are small hydrophobic molecules of coenzyme Q that diffuse more rapidly. Water-soluble molecules of cytochrome c circulate within the intermembrane space (represented at the top of the illustration). The mitochondrial matrix would be at the bottom of the illustration where the Roman numerals are shown.

If considered as a set, the electron transport system (ETS) consists of four (I-IV) complexes plus many molecules of coenzyme Q and cytochrome c. Complex V takes part in 'oxidative phosphorylation' -- the final step. The details of this illustration are too small to read at this resolution but each will be fully drawn out in the following tutorials.

Complex I

Complex I, also called NADH dehydrogenase, has a binding site for NADH that hangs into the matrix. Two electrons are transferred into the complex while the hydrogen ion and the oxidized NAD+ are released into the matrix. The electron pair is passed between several molecules within the complex to the complex surface buried within the inner membrane. There is a bonding site there for coenzyme Q.

Existing within the hydrophobic interior of the inner membrane are small mobile molecules of coenzyme Q. As common with electron transporters these have 'tag along' hydrogen nuclei (H+). These hydrogen ions are picked up directly from the matrix pool and join the electron pair to fully reduce coenzyme Q to QH2. As this is occurring there is a conformational change -- possibly forming four channels -- that 'pumps' four (4) hydrogen ions from the matrix pool into the intermembrane space.

The key points are:

  • One (1) NADH is oxidized and donates:

  • Complex II

    Complex II, also called succinate dehydrogenase , contains the actual enzyme we saw in step 6 of Krebs' cycle. It's binding site for succinate is on the matrix side of the membrane and the transporter (FAD) is actually part of the complex itself. The electrons from the succinate reduce FAD to FAD-2 while the hydrogen ions and fumarate are released into the matrix.

    A FADH2, from within the complex, is oxidized producing a pair of hydrogen ions to complete the reduction of the newly-formed FAD-2 thus regenerating itself. The FAD will accept the next pair of electrons that are received. The electron pair is transferred to a coenzyme Q attached to the surface of the complex to form Q-2. Finally, a pair of hydrogen ions from the pool in the matrix completes the reduction of the coenzyme to QH2.

    The key points are:

  • One succinate molecule is reduced to donate:

  • Complex III

    Initial reactants for this enzyme complex are coenzyme QH2 molecules (shown at the left side of the complex). These have been accumulating in the hydrophobic interior of the inner membrane due to the activities of complexes I and II.

    Complex III, also called coenzyme Q - cytochrome c reductase. simultaneously reduces both coenzyme Q (illustrated at the right of the complex) and cytochrome c (illustrated at the top of the complex). Cytochrome c molecules are numerous and located within the IMS. Like complexes I and II, complex III also reduces coenzyme Q to QH2 (at the right of the complex).

    To understand what occurs we must follow one QH2 at a time. Inspect the top QH2 at the left of the complex. Notice it contacts the surface of the complex to release a pair of electrons (red) to the complex and the pair of hydrogen ions (red) into the IMS.

    Following the electrons (red arrows) notice that:

    Now follow the products (blue) of the second QH2. A second pair of hydrogen ions is added to the intermembrane space. Another electron (blue) reduces a second cytochrome c. The second electron (blue) is transferred to the half-way reduced coenzyme Q plus a pair of hydrogen ions from the matrix completes the formation of a molecule of QH2. It is released from the surface of complex III to return to the hydrophobic interior of the membrane -- and complexes I and II.

    The hydrogen ion 'pump' of this complex is indirect. In complexes I (and IV) hydrogen ions are physically moved from the matrix to the space. In this case two pairs of hydrogen ions are moved out of the interior of the inner membrane while one pair is moved from the matrix into the interior of that membrane. *This is equivalent to moving a pair from the matrix to the space. Complex II is credited with pumping one pair of hydrogen ions into the intermembrane space instead of two.

    The key points are:

  • For each pair of QH2 oxidized:
  • :

    Complex IV

    Complex IV, also called cytochrome c oxidase (COX) . will oxidize the reduced coenzyme c molecules produced by complex III returning the oxidized forms to the intermembrane space -- and to complex III. Simultaneously, one molecule of molecular oxygen (O2) from the matrix is taken into the complex. Current research indicates four electrons, donated by four cytochrome c molecules, break the double-bond between the oxygen atoms immediately forming two oxygen ions (O-2). The mechanism is unclear as to how four hydrogen ions from the matrix are simultaneously pumped into the intermembrane space. Each oxygen ion receives a pair of hydrogen ions from the matrix to form water. Oxygen is known as 'the final electron acceptor' reducing molecular oxygen to water.

    The key points are:

  • For every four (4) cytochrome c molecules that are oxidized the following occurs:

  • Shuttles

    There are two (2) NADH molecules produced in the cytoplasm during glycolysis. They cannot cross the inner membrane. In order to utilize their potential to donate electrons they use one of two shuttle systems. These shuttles are self replenishing so as not to run out of participating molecules.

    Malate-Aspartate Shuttle

    The illustration shows the inner membrane placed vertically with the IMS on it's left and the matrix on it's right. There are two antiports (exchangers) embedded in the inner mitochondrial membrane that swap molecules across the membrane.

    The overall big picture of all these reactions looks like two circles; a smaller one inside a larger one. The arrows of the larger outer circle go clockwise while those of the inner one go counter-clockwise.

  • On the IMS side of the outer circle, aspartate is converted to oxaloacetate that is converted to malate. On the matrix side the same conversions occur in the opposite direction.
  • On the IMS side of the small inner circle ,alpha-ketoglutarate is converted to glutamate while the opposite conversions occur in the matrix.
  • Notice NADH + H+ in the IMS reducing oxaloacetate to malate. Malate now is the bearer of the electrons and hydrogens with which we are concerned. To move these electrons and hydrogens -- within malate --to the matrix there must be an alpha-ketoglutarate bound to the antiport molecule. Only when both molecules --malate and alphaketoglutarate -- are attached will the antiport simultaneously move them to opposite sides of the membrane. Once in the matrix the enzyme for step 8 of Krebs' cycle converts the malate to oxaloacetate releasing NADH and H+. Mission accomplished. Now NADH can donate it's electrons to complex I of the ETS.

    The matrix loses a molecule of alpha-ketoglutarate, an important substrate in Krebs' cycle, during the above event. To compensate for this loss a new molecule is formed when oxaloacetate (formerly malate) reacts with glutamate that has been transferred in from the IMS. During this reaction aspartate is formed. An antiport for glutamate and aspartate insures a continuing supply of glutamate for future matrix reactions.

    Inspection of the IMS side of the membrane shows that alpha-ketoglutarate is converted to glutamate to insure it's continuing supply. Since it is moved to the matrix by an antiport there must also be a continuing supply of its 'partner' aspartate. Aspartate is produced from oxaloacetate as mentioned earlier. Also notice that once in the IMS the aspartate is converted to oxaloacetate to continue the outer-circle reactions. A lot of work to move regenerate a single NADH on the matrix side of the membrane.

    Glycerol Phosphate Shuttle

    This shuttle system is primarily found in brown fat and infants. A NADH produced in the cytoplasm during glycolysis can react with DHAP reducing it to glycerol-3-phosphate that now carries the electrons and hydrogens. The enzyme complex embedded in the inner membrane (lower right) contains the enzyme FAD-dependent glyceolphosphate dehydrogenase. There is a binding site for glycerol-3-phosphate on it's IMS side. The enzyme oxidizes this reactant while reducing FAD that is an integral part of the complex. Notice that FAD accepts only the electron pair (blue) ; the hydrogen ion pair (red) is set free in the IMS. The FAD-2 then reduces a coenzyme Q within the hydrophobic inner membrane ; this picks up a pair of hydrogen ions from the matrix to form QH2 -- now available to react with complex III of the ETS.

    The difference between using this shuttle as opposed to the previous shuttle is that the previous one releases the electron pair to complex I -- early in the ETS -- while this shuttle releases the electrons to complex III.

    Shuttle Summary

    The point of knowing which shuttle is used to capture the electron-transferring power of cytoplasmic NADH molecules comes into play when trying to determine the number of ATP molecules formed as a result of events occurring in the ETS. This will be discussed after the following section. The key point is that if the malate-aspartate shuttle is used there is a NADH regenerated from a NADH. If the glycerol phosphate shuttle is used a QH2 is produced. NAD enters the ETS at complex I so that it's electron pair will pass through all three proton-pumping complexes. However, QH2 gives it's electron pair to complex III bypassing the proton-pumping power of complex I.

    Oxidative Phosphorylation

    This group of reactions utilizes the hydrogen ion gradient across the inner membrane to produce ATP. The term 'oxidative' refers to the fact that the hydrogen ions were concentrated on the IMS side of the membrane due to the oxidation of the reduced transporters previously formed. 'Phosphorylation' refers to the addition of a phosphate group to ADP forming ATP.

    Complex V

    The illustration shows that this complex has two major parts; that embedded within the membrane (between dotted lines) and that suspended into the matrix (lower part of illustration). The embedded part has a central shaft that extends into the matrix part. This complex is actually a motor -- it moves! The embedded part rotates -- staff included. The outer portion of the matrix part is stationary but the shaft within it does move. The 'fuel' for the rotation is the diffusion of hydrogen ions, along their electrochemical gradient (proton motive force), from the IMS (top of illustration) to the matrix.

    Current literature reports ten (10) proteins encircling the shaft within the membrane in human mitochondria. When there are ten, research suggests that a set of three (3) hydrogen ions, diffusing through a separate protein 'channel' will turn the membrane portion 120 degrees. It appears that the number of proteins encircling this part of the shaft determines the number of hydrogen ions required to produce this rotation.

    There are three (3) sets of protein pairs (alpha and beta, not labeled) that fit side-by-side surrounding the shaft in the matrix part. The rotation of the shaft causes shape changes of the binding sites in these pairs.

    The shaft between the three unit pairs has three 'spikes' arranged so that one contacts each pair simultaneously (see illustration). With each 120 degree rotation the spikes rotate to contact the adjacent alpha/beta pair. Another 120 degree rotation moves them to the next pair, etc. When spike #1 contacts a unit physical conformation of the pair changes to allow an ADP and inorganic phosphate to enter. When spike #2 contacts this pair the bonding sites changes again to bring the reactants into contact so ATP can be synthesized. Contact with spike #3 separates the pair to release ATP and the return of spike #1 alters the conformation to accept the next pair of reactants.

    Each alpha/beta pair is 'one-step-behind' the one before it thus there is an ATP released with each 120 degree turn of the shaft. The stoichiometry (i.e., relation between the amount of two things) between the diffusion of three (3) hydrogen ions is the production of one (1) ATP -- 3H+:1ATP


    ADP is formed in the cytoplasm and has too great a negative charge to pass through the inner mitochondrial membrane. Likewise, ATP formed within the matrix can not pass through either for the same reason. Many copies of a transporter called adenosine nucleotide translocase (ANT), embedded in the inner membrane, serve the exchange one molecule of ADP for one molecule of ATP.

    The mechanism for the exchange involves conformational changes. At any time a bonding site exists on one side or the other but not both simultaneously as implied by the illustration. A site on the matrix side will bind ATP. Once bound the molecule changes to evert the ATP to the IMS. After that a new site for ADP forms on the IMS side. When an ADP binds the eversion takes place in reverse. Take note that the exchange creates a change in the charge (voltage) difference across the membrane.

    Inorganic phosphate is also in the cytoplasm but cannot pass through the membrane due to it's charge. Multiple copies of the translocator phosphate translocase, embedded in the membrane, relocate one inorganic phosphate plus a hydrogen ion from the IMS into the matrix. In this case both substrates move in the same direction. This is termed 'symport'; both substrates must be bound to activate the 'pump'. The driving force is the hydrogen ion gradient favoring transport from the IMS to the matrix. Current literature favors counting the transfer of this hydrogen ion into the matrix along with the three moving through complex V when tabulating the number of ATPs formed.

    ATP Synthesis

    Synthesis of ATP required the movement of hydrogen ions along a gradient moving them from the IMS to the matrix. This gradient is produced by the pumping of hydrogen ions into the IMS as described above. We now believe it requires the movement of four (4) hydrogen ions -- three through complex V plus one via the phosphate/hydrogen ion symport -- back into the matrix to synthesize one (1) ATP. By tabulating the number of hydrogen ions moved into the IMS per glucose decomposed we can calculate the hypothetical number of ATP molecules produced per glucose molecule decomposed.

    With new insight into the structure and function of the ETS complexes, plus the role of translocators, our calculations of the number of ATP molecules synthesized per glucose molecule has changed. Many students are still taught that 36-38 ATPs are formed per glucose. The calculations leading to these values are based on a an outdated understanding of the mechanisms involved in the ETS. We currently believe 30-32 is the range of ATPs produced as explained below.

    Source of Hydrogen Ions

    FADH2 in matrix

    Two molecules of FADH2 are produced in the matrix during Krebs' cycle. These transfer their electrons to complex II where each generates a molecule of coenzyme QH2. Each reduced coenzyme transfers it's electrons to complex III and the electron transfer from that point on only adds six (6) hydrogen ions to the IMS. Because it requires four (4) hydrogen ions to generate one (1) ATP then each FADH2 contributes sufficient hydrogen ions to produce one and a half (6/4=1.5) ATP molecules.

    NADH in matrix

    Each NADH in the matrix will add 10 hydrogen ions to the IMS. Because it requires four (4) hydrogen ions to generate one (1) ATP then each NADH contributes sufficient hydrogen ions to produce two and a half (10/4=2.5) ATP molecules.

    NADH in cytoplasm

    This is where the variation in numbers of ATPs formed comes into play. Two NADH molecules are formed in the cytoplasm. Each must use a shuttle to deliver their electrons to the ETS. Which shuttle is used is up to chance.

    1. Use of the malate-aspartate shuttle delivers one NADH to the matrix where it adds ten (10) hydrogen ions to the IMS.
    2. AND/OR
    3. Use of the glycerol phosphate shuttle transfers a pair of electrons to coenzyme QH2. From here, electrons are transferred to complex III and the electron transfer from that point on only adds six (6) hydrogen ions to the IMS.

    Final Calculations

    Regardless of which shuttle is used by the two (2) cytoplasmic NADH molecules, there are eight (8) NADH molecules that will produce (8x2.5=20) twenty(20) ATP molecules. There are also two (2) FADH2 molecules that will produce (2x1.5=3) three (3) ATP molecules. This totals twenty-three (23).

    Substrate-level Phosphorylation

    There were two ATP molecules produced directly by during glycolysis. There were two more NTP (translate ATP) molecules produced during Krebs' cycle. Adding these four (4) to the 26-28 range calculated for ATP molecules produced by oxidative phosphorylation gives a final range of 30-32 per molecule of glucose broken down.