The respiratory chain is embedded in cristae of the inner membrane.

Spontaneous electron transfer through respiratory chain complexes I, III, & IV is coupled to H+ ejection from the matrix to the intermembrane space. Because the outer membrane contains large channels, protons in the intermembrane space equilibrate with the cytosol. Respiration-linked pumping of protons out of the mitochondrial matrix conserves some of the free energy of spontaneous electron transfers as potential energy of an electrochemical H+ gradient.

Electron transfer from NADH to O2 involves multi-subunit inner membrane complexes I, II, III, & IV, plus coenzyme Q and cytochrome c. Within each complex, electrons pass sequentially through a series of electron carriers.

The composition of each of the respiratory chain complexes is shown below:




No. of Proteins

Prosthetic Groups

Complex I

NADH Dehydrogenase


FMN, 9 Fe-S centers

Complex II

Succinate-CoQ Reductase


FAD, cyt b560, 3 Fe-S centers

Complex III

CoQ-cyt c Reductase


cyt bH, cyt bL, cyt c1, Fe-SRieske

Complex IV

Cytochrome Oxidase


cyt a, cyt a3, CuA, CuB






A total of 10 protons are ejected from the mitochondrial matrix per 2 electrons transferred from NADH to oxygen via the respiratory chain. The H+/e- ratio for each respiratory chain complex will be discussed separately.



Complex I (NADH Dehydrogenase) transports 4H+ out of the mitochondrial matrix per 2e- transferred from NADH to coenzyme Q.  Complex I catalyzes oxidation of NADH, with reduction of coenzyme Q

NADH + H+ + Q NAD+ + QH2


Lack of high-resolution structural information for the membrane domain of complex I has hindered elucidation of the mechanism of H+ transport through this complex. Direct coupling of transmembrane proton flux and electron transfer is unlikely, because the electron-transferring prosthetic groups, FMN and iron-sulfur centers, are all located in the peripheral domain of complex I. Thus it is assumed that protein conformational changes are involved in H+ transport, as with an ion pump.

Succinate Dehydrogenase of the Krebs Cycle  is also called complex II or Succinate-CoQ Reductase.


FAD is the initial electron receptor.  FAD is reduced to FADH2 during oxidation of succinate to fumarate.  FADH2 is then reoxidized by transfer of electrons through a series of three iron-sulfur centers to Coenzyme Q, yielding QH2.



X-ray crystallographic analysis of E. coli complex II indicates a linear arrangement of electron carriers within complex II, consistent with the predicted sequence of electron transfers:

FAD FeScenter 1 FeScenter 2 FeScenter 3 CoQ


Complex III (bc1 complex): H+ transport in complex III involves coenzyme Q (CoQ). The "Q cycle" depends on the mobility of CoQ within the lipid bilayer. There is evidence for one-electron transfers, with an intermediate semiquinone radical state.


Q Cycle:








  • Electrons enter complex III via coenzyme QH2, which binds at a site on the positive side of the inner mitochondrial membrane, adjacent to the intermembrane space.
  • QH2 gives up one electron to the Rieske iron-sulfur center (Fe-S).
    Fe-S is reoxidized by transfer of the electron to cytochrome c1, which passes it out of the complex to cytochrome c. 
    The loss of one electron from QH2 would generate a semiquinone radical, shown here as Q-, although the semiquinone might initially retain a proton as QH.
  • A second electron is transferred from the semiquinone to cytochrome bL (heme bL), which passes it across the membrane via cytochrome bH (heme bH) to another CoQ bound at a site on the matrix side of the membrane.
    The fully oxidized CoQ, generated as the second electron is passed to the b cytochromes, may then dissociate from its binding site adjacent to the intermembrane space.
  • Accompanying the two-electron oxidation of bound QH2, 2H+ are released to the intermembrane space.


In an alternative mechanism that has been proposed, the two electron transfers from QH2 to Fe-S & cyt bL may be essentially simultaneous, eliminating the semiquinone intermediate.

It takes 2 cycles for CoQ, bound at the site near the matrix side of the membrane, to be reduced to QH2, as it accepts 2 electrons from the b hemes and 2 H+ are extracted from the matrix compartment. In 2 cycles, 2 QH2 enter the pathway, and one is regenerated.

Overall reaction catalyzed by complex III, including net inputs and outputs of the Q cycle: 
            QH2 + 2H+(matrix side) + 2 cyt c (Fe3+) Q + 4H+(outside) + 2 cyt c (Fe2+)

Per 2e- transferred through the complex to cytochrome c, 4H+ are released to the intermembrane space. While 4H+ appear outside per net 2e- transferred in 2 cycles, only 2H+ are taken up on the matrix side. In respiratory chain complex IV (see below), there is a similarly uncompensated uptake of protons from the matrix side (4H+ per O2 or 2 per 2e-). Thus there are 2H+ per 2e- that are effectively transported by a combination of complexes III & IV. They are listed with complex III in diagrams depicting H+/e- stoichiometry.

 The b hemes are positioned to provide a pathway for electron transfer across the membrane.

The protein domain with attached Rieske iron-sulfur center (labeled Fe-S) has a flexible link to the rest of the complex. At right, the iron-sulfur center protein is colored green. The iron-sulfur center changes position during electron transfer. After Fe-S extracts an e- from QH2, it moves closer to heme c1 (cytochrome c1) to which it transfers the e-. After the first electron transfer from QH2 to Fe-S, the CoQ semiquinone is postulated to shift position within the Q-binding site, moving closer to its electron acceptor, heme bL. This would help to prevent transfer of the second electron from the semiquinone to Fe-S.





Complex IV (Cytochrome Oxidase): Electrons are donated to complex IV, one at a time, by cytochrome c, which binds from the intermembrane space. Each electron passes via CuA and heme a to the binuclear center, buried within the complex, that catalyzes oxygen reduction:
                                                4e- + 4H+ + O2 → 2H2O

Protons utilized in this reaction are taken up from the matrix compartment.

In addition to the protons utilized in the reduction of O2, there is electron transfer-linked transport of 2H+ per 2e- (4H+ per 4e-) from the matrix to the intermembrane space.

Structural and mutational studies indicate that protons pass through complex IV via chains of groups subject to protonation/deprotonation, called "proton wires." These consist mainly of chains of buried water molecules, along with amino acid side-chains, and propionate side-chains of the hemes.

Separate H+-conducting pathways link each side of the membrane to the buried binuclear center where O2 reduction takes place. These include two proton pathways, designated "D" and "K" (named after constituent Asp and Lys residues) extending from the mitochondrial matrix to near the binuclear center deep within complex IV.
 A switch mechanism controlled by the reaction cycle is proposed to effect transfer of a proton from one half-wire (half-channel) to the other. There cannot be an open pathway for H+ completely through the membrane, or oxidative phosphorylation would be uncoupled. (Pumped protons would leak back;). The process of switching may involve conformational changes, and oxidation/reduction-linked changes in pKa of groups associated with the catalytic metal centers.


The ATP synthase, which is embedded in cristae of the inner mitochondrial membrane, includes the following major subunits:

         F1 - the catalytic subunit, made of 5 polypeptides with stoichiometry a3b3gde.

         Fo - a complex of integral membrane proteins that mediates proton transport. 

The F1Fo complex couples ATP synthesis to H+ transport into the mitochondrial matrix. Transport of at least 3H+ per ATP synthesized is required, as estimated from a comparison of the following:


The Chemiosmotic Theory of oxidative phosphorylation, for which Peter Mitchell received the Nobel prize is summarized in the diagram.

The Chemiosmotic Theory states that coupling of electron transfer to ATP synthesis is indirect, via a H+ electrochemical gradient:


1.      Respiration: Spontaneous electron transfer through complexes I, III, and IV is coupled to non-spontaneous H+ ejection from the mitochondrial matrix. H+ ejection creates a membrane potential (DY, negative in the matrix) and a pH gradient (DpH, alkaline in the matrix).

2.      F1Fo ATP Synthase: Non-spontaneous ATP synthesis is coupled to spontaneous H+ transport into the matrix compartment. The pH and electrical gradients created by respiration are together the driving force for H+ uptake.
Return of protons to the matrix via Fo "uses up" the pH and electrical gradients.

ATP produced in the mitochondria must exit to the cytosol to be used by transport pumps, kinases, etc. ADP and Pi, arising from ATP hydrolysis in the cytosol, must re-enter the mitochondria to be converted again to ATP. 

Two carrier proteins in the inner mitochondrial membrane are required for this metabolic cycle. The outer membrane is considered to be not a permeability barrier. The large VDAC channels in the outer membrane are assumed to allow passage of adenine nucleotides and Pi


1.      The Adenine Nucleotide Translocase (ADP/ATP carrier) is an antiporter that catalyzes exchange of ADP for ATP across the inner mitochondrial membrane (p. 496). At cellular pH, ATP has four negative charges, while ADP has 3 negative charges. ADP3-/ATP4- exchange is driven by, and uses up, the membrane potential generated by respiration (one charge per ATP).

2.      Phosphate reenters the mitochondrial matrix with H+, by an electroneutral symport mechanism. Pi entry is driven by and uses up the pH gradient (equivalent to one mole of H+ per mole of ATP).

Thus the equivalent of one mol of H+ enters the matrix with ADP/ATP exchange and Pi uptake. Assuming transport of 3 mol H+ by F1Fo, a total of 4H+ would enter the mitochondrial matrix per ATP synthesized.

The phenomenon of respiratory control is the subject of important study. An oxygen electrode may be used to record [O2] in a closed vessel. Electron transfer, e.g., from NADH to O2, is monitored by recording the rate of disappearance of O2.

At right is an idealized representation of an oxygen electrode recording while mitochondria respire in the presence of Pi, along with an electron donor (e.g., succinate, or a substrate of a reaction that will generate NADH).

The dependence of respiration rate on availability of ADP, the substrate for the ATP Synthase, is called respiratory control.  The respiratory control ratio is the ratio of slopes after and before ADP addition (b/a).  The P/O ratio is the moles of ADP added, divided by the moles of O consumed (based on c) while phosphorylating the added ADP.


Chemiosmotic explanation of respiratory control: 

Electron transfer is obligatorily coupled to H+ ejection from the matrix. Whether this coupled reaction is spontaneous depends on the pH and electrical gradients.


Free energy change

e- transfer (e.g., NADH to O2)

a negative value*

H+ ejection from the matrix

a positive value that varies with the H+ gradient**

e- transfer coupled to H+ ejection

algebraic sum of the above

*DGo' = - nFDEo' = -218 kJ/mol, for transfer of 2 e- from NADH to O2.

** For ejection of one H+ from the matrix:
DG = RT ln ([H+]cytosol/[H+]matrix) + F DY = 2.3 RT (pHmatrix - pHcytosol) + F DY

In the absence of ADP, H+ cannot flow back to the matrix through Fo. The pH and electrical gradients (DpH & DY) are maximal. As respiration with outward H+ pumping proceeds, the free energy change for H+ ejection (positive DG) increases and approaches the magnitude of that for electron transfer (negative DG). When the coupled reaction becomes non-spontaneous, respiration stops. This is referred to as a static head. In fact there is usually a low rate of respiration in the absence of ADP, attributed to H+ leaks. 

When ADP is added, H+ enters the matrix via Fo, as ATP is synthesized. This reduces the pH and electrical gradients. DG of H+ ejection decreases. The coupled reaction of electron transfer with H+ ejection becomes spontaneous. Respiration resumes or is stimulated.

Uncoupling reagents (uncouplers) are lipid-soluble weak acids.  For example, H+ (shown in red) can dissociate from the hydroxyl group of the uncoupler dinitrophenol.  Uncouplers dissolve in the membrane, and function as carriers for H+.

Uncouplers block oxidative phosphorylation by dissipating the H+ electrochemical gradient.



Protons pumped out are carried by the uncoupler back into the mitochondrial matrix, preventing development of a pH or electrical gradient.



With an uncoupler present there is no pH or electrical gradient. DG for H+ ejection is zero, and DG for e- transfer coupled to H+ ejection is maximal (spontaneous). Respiration proceeds in the presence of an uncoupler, whether or not ADP is present.  Since DG for H+ flux is zero in the absence of a H+ gradient, and hydrolysis of ATP is spontaneous, the ATP Synthase reaction runs backward in the presence of an uncoupler.

An uncoupling protein (also called thermogenin) is produced in brown adipose tissue of newborn mammals and hibernating mammals (see p. 834-835). This protein of the inner mitochondrial membrane functions as a H+ carrier.


The uncoupling protein blocks development of a H+ electrochemical gradient, thereby stimulating respiration. The free energy change associated with respiration is dissipated as heat. This "non-shivering thermogenesis" is costly in terms of respiratory energy unavailable for ATP synthesis, but it provides valuable warming of the organism.

Respiratory chain inhibitors include the following:
    Rotenone (a common rat poison) blocks electron transfer in complex I.
    Antimycin A blocks electron transfer in complex III.
    Cyanide and carbon monoxide inhibit complex IV.
Inhibition at any of these sites will block electron transfer from NADH to oxygen.

The open axial ligand position of the iron atom in heme a3 makes it susceptible to binding each of the following inhibitors: CN-, CO, and the radical signal molecule NO (nitric oxide).

NO may regulate cellular respiration through its inhibitory effect, and can induce a condition comparable to hypoxia.


F1Fo ATP Synthase of mitochondria, chloroplasts, and bacteria is represented schematically at right. When the electrochemical H+ gradient is favorable, F1Fo catalyzes ATP synthesis coupled to spontaneous H+ flux toward the side of the membrane where F1 protrudes. E.g., in mitochondria, the pH and electrical gradients drive H+ transport from the intermembrane space to the matrix compartment.

If no membrane potential or pH gradient exists to drive the forward reaction, the Keq favors the reverse reaction, ATP hydrolysis (ATPase activity).




Inhibitors of F1Fo, that block H+ transport coupled to ATP synthesis or hydrolysis, include:

  • oligomycin, an antibiotic
  • DCCD (dicyclohexylcarbodiimide), a reagent that reacts with carboxyl groups in hydrophobic environments, forming a covalent adduct.



Viewed by electron microscopy with negative staining, the ATP synthase appeared as "lollipops" on the inner mitochondrial membrane, facing the matrix.  Higher resolution cryo-electron microscopy later showed each lollipop to have two stalks.

Roles of major subunits were determined in studies of submitochondrial particles (SMP). If mitochondria are treated with ultrasound, the inner membrane breaks and reseals as vesicles, with F1 on the outer surface. Since F1 of intact mitochondria faces the interior matrix space, these SMP are said to be inside out.


         F1, the lollipop head, when extracted from SMP, catalyzes ATP hydrolysis (the spontaneous reaction in the absence of an energy input). Thus F1 contains the catalytic domain(s).

         After removal of F1, the SMP membrane containing Fo is leaky to H+. Adding back F1 restores the normal low permeability to H+. Thus it was established that Fo includes a "proton channel."

         Either oligomycin or DCCD blocks the H+ leak in membranes depleted of F1. Thus oligomycin and DCCD inhibit the ATP Synthase by interacting with Fo.

The subunit composition of the ATP Synthase was first established for E. coli, which has an operon that encodes genes for all subunits. Stalk subunits were classified initially as being part of either F1 or Fo, based on whether they co-purified with extracted F1.

  • F1 subunits, as originally classified, were named with Greek letters in order of decreasing molecular weight. They are present in stoichiometry a3, b3, g, d, e.
    • The a and b subunits (513 and 460 amino acid residues in E. coli), are homologous to one another. Looking down at the membrane, a & b subunits alternate around a ring. (The g subunit is discussed below.)
    • There are three nucleotide-binding catalytic sites, located at ab interfaces but predominantly involving residues of the b subunits.
    • Each a subunit contains a tightly bound ATP, but is inactive in catalysis.
    • Adenine nucleotides bind to both a and b subunits with Mg++.


Mammalian mitochondrial F1Fo is slightly more complex than the bacterial enzyme, with a few additional subunits. Also, since names were assigned based on apparent molecular weights, some subunits were given different names in different organisms.

There is evidence that the ATP Synthase (F1Fo) may form a complex with the adenine nucleotide translocase (ADP/ATP antiporter) and the phosphate carrier (Pi/H+ symporter). This complex has been designated the ATP Synthasome.