Lecture 6 Notes


  1. Stoichiometries of Oxidative Phosphorylation
    1. Redox energy (ΔE), protonmotive force (Δp), and ΔG
    2. Stoichiometry of the Electron Transport Chain
    3. Stoichiometry of the ATP Synthase

Stoichiometries of Oxidative Phosphorylation

    We know that passage of electrons down the ETS is coupled to electrogenic proton ejection and that ATP synthesis is driven by electrophoretic proton back-flux through the F1,F0 ATPase of mitochondria.  The stoichiometries of oxidative phosphorylation answer the quantitative questions, how many protons are pumped per pair of electrons moving through all or part of the ETS?  How many protons are required to synthesize one mole of ATP?

    The quantities commonly dealt with in the literature are H+/O, H+/P, and P/O ratios.  Here, we will define these quantites and explore their values.

Redox energy (ΔE), protonmotive force (Δp), and ΔG


    Recall from Lecture 3a that a charge (electron or ion) moving through a voltage is a form of work, and a charge having the potential to move through a voltage is a form of energy.  The general conversion between electrical force and energy is:

                    ΔG = znFΔV                (1)

where ΔG is in J/mol, z is the valence, n is the number of charges moved, and F is the Faraday (F = 96485.3415 Coulombs).

    We can apply this equation to the electron transport chain:

                    ΔG = -ne FΔE                (2)

where the minus sign arises from the valency of an electron.  Recall that the redox energy, ΔE, for NADH to O is about 1.16 V and that 2 electrons pass to O.  Therefore,

            ΔG = -2 x 96.485 x 1.16 =  -216 kJ/mol

The energy of electron transport is transduced to the energy of an electrochemical gradient of protons ΔμH+.  In general, there will be some losses in this energy transfer, so

            nH+ΔμH+  ≤ ΔG                        (3)

where nH+ = the H+/O stoichiometry of the electron transport chain.

Recall that protonmotive force is defined

            Δp   =   - ΔμH+/F                         (4)


Therefore, combining Eqns 2-4,

            nH+Δp  ≤ 2 ΔE                         (5)

Stoichiometry of the Electron Transport Chain

    We can use values of Δpo from the mitochondrial “battery curve” to estimate possible values of the  H+/O stoichiometry.   Thus, for NADH to O, 2 ΔE   ~   2.32 V, and for succinate to O,  2 ΔE   ~   1.56 V.   In both cases, the measured Δpo is about 208 mV.  Evidently, nH+   ≤ 11 for NADH to O, and  nH+   ≤ 7 for succinate to O.

    During the 1970-1987 period, after the Chemiosmotic Theory was accepted, many laboratories set out to determine these fundamental quantities.  (See Fig. 4.6 in Nicholls and Ferguson for the experimental technique used by Mitchell and others). As you have heard, Peter Mitchell advocated an H+/O stoichiometry of 6 for NADH to O and 4 for succinate to O.  Other laboratories, beginning with Pressman and Azzone and going on with Lehninger and coworkers, found much higher values by direct experiment.  Interestingly, the consensus values found in most textbooks today are 10 and 6.  However, Andrew Beavis, writing from my laboratory in 1987, has made a very persuasive case that the correct values are 11 and 7 [1, 2].

Stoichiometry of the ATP Synthase

    ATP synthesis is driven by Δp, and we may write

        nP Δp   ≥ ΔGP /F                            (6)

where nP is the H+/P stoichiometry (number of protons required to synthesize one mole of ATP) and  ΔGP is the “phosphorylation potential”, the free energy of ATP synthesis.  
When ATP is synthesized in the matrix, it must exit via the adenine nucleotide translocase (ANT), which catalyzes 1:1 exchange of ATP for external ADP with movement of one anionic charge outward.  This electrophoretic transporter therefore requires an additional proton to be pumped out electrogenically.  (The proton is restored by influx of phosphate, essentially as phosphoric acid).  Consequently, if we consider cytosolic ΔGP, nP refers to the sum of the protons ejected to drive ATP synthesis and the proton ejected to drive ATP/ADP exchange.

If we take  ΔGP (cytosolic) = 62 kJ/mol and Δp ~ 200 mV, it follows that

        nP    ≥   3.2                                  (7)


The consensus view has been that   nP   =   4.    

    We may now combine these ratios to obtain a quantity that can be estimated independently, namely the P/O ratio.

    P/O    =    nH+ / nP

The consensus numbers follow from 10 and 6 for nH .  That is, the P/O ratio for NADH is 2.5 and that for succinate is 1.5.   Interestingly, Beavis and Lehninger measured P/O ratios independently and obtained values of 2.75 and 1.75 [3, 4].  These values are of course consistent with Beavis’ H+/O stoichiometries of 11 and 7, described above.

(Albert Lehninger, who was my Professor of Biochemistry in Medical School, died on March 4, 1986).


    1.    Beavis, A. D. (1987) Upper and lower limits of the charge translocation stoichiometry of mitochondrial electron transport. J Biol Chem 262, 6165-73.
    2.    Beavis, A. D. (1987) Upper and lower limits of the charge translocation stoichiometry of cytochrome c oxidase. J Biol Chem 262, 6174-81.
    3.    Beavis, A. D. and Lehninger, A. L. (1986) The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation. Eur J Biochem 158, 315-22.
    4.    Beavis, A. D. and Lehninger, A. L. (1986) Determination of the upper and lower limits of the mechanistic stoichiometry of incompletely coupled fluxes. Stoichiometry of incompletely coupled reactions. Eur J Biochem 158, 307-14.