📒 Wilson 2014

Mitochondrial cytochrome c oxidase: mechanism of action and role in regulating oxidative phosphorylation1



Mitochondrial cytochrome c oxidase (complex IV) is uniquely positioned to act as the control unit that sets and maintains metabolic homeostasis

@ pH 7.2

  • cyt c: 235mV
  • cyt a: 210mV
  • Cu: 225mV & 350mV
  • cyt a3: 375mV

The oxygen was reduced was between the two metal atoms and that reduction proceeded through an intermediate with peroxide bridged between the iron of cytochrome a3 and the high-potential copper atom

This paper presents a mechanism (model) for oxygen reduction by cytochrome c oxidase and critically evaluates the chemical and thermodynamic requirements of each step of oxygen reduction


Mitochondrial Data

  • Measurements of isolated mitochondria are taken from published paper
  • Oxygen reduction by cytochrome c oxidase has strict thermodynamic constraints
    • Hydroperoxyl radical (HO2) formation @ pH 7: -330mV ~ NAD/NADH
    • Peroxide (H2O2) formation: 300mV ~ cyt c
    • The site at which oxygen is reduced to water needs to have a potential near 0.6 V

Oxygen Reduction by Cytochrome c Oxidase

Reaction 1

Oxidation of Cyt c and reduction of copper $$ \mathrm{a}{3}^{3+}-\mathrm{Cu}^{2+}+\mathrm{c}^{2+} \leftarrow \frac{\mathrm{k1}=8 \times 10^{9}}{\mathrm{k1r} =8 \times 10^{7}} \rightarrow \mathrm{c}^{3+}+\mathrm{a}{3}^{3+}-\mathrm{Cu}^{1+}(1) $$

  • In isolated mitochondria, the equilibrium constant for this reaction is near 100

Reaction 2

Binding of oxygen $$ \mathrm{a}{3}^{3+}-\mathrm{Cu}^{1+}+\mathrm{O}{2} \leftarrow \frac{\mathrm{k} 2=6 \times 10^{8}}{\mathrm{k} 2 \mathrm{r}=10} \rightarrow \mathrm{a}{3}^{3+}-\mathrm{Cu}^{1+}-\mathrm{O}{2}(2) $$

  • Oxygen binds with high affinity (Kd <10−7 M), and the off constant has been estimated to be less than 10 / s

Reaction 3

Exchange of electron between the metal centers $$ \mathrm{a}{3}^{3+}-\mathrm{Cu}^{1+}-\mathrm{O}{2}+\mathrm{H}^{+} \leftarrow \frac{\mathrm{K} 3=2 \times 10^{6} \mathrm{M}^{-1}}{} \rightarrow \mathrm{O}{2}-\mathrm{a}{3}^{2+}-\mathrm{Cu}^{2+}(3) $$

  • a proton is taken up when cytochrome a3 is reduced (so it is pH-dependent)
  • equilibrium constant, K3: 2 × 10^6 M−1 or 0.2 at pH 7.0

Reaction 4

Formation of peroxide (irreversible, +1270mV) $$ a_{3}^{3+}-\mathrm{Cu}^{1+}-\mathrm{O}_{2}+\mathrm{c}^{2+} \frac{\mathrm{k} 4 \mathrm{a}=2.5 \times 10^8}{} \rightarrow \mathrm{c}^{3+}+\mathrm{a}_{3}^{3+}-\mathrm{O}^{2-}-\mathrm{Cu}^{2+}(4 \mathrm{a}) $$ $$ \mathrm{O}_{2}-\mathrm{a}_{3}^{2+}-\mathrm{Cu}^{2+}+\mathrm{c}^{2+} \frac{\mathrm{k} 4 \mathrm{b}=8 \times 10^{7}}{} \rightarrow \mathrm{c}^{3+}+\mathrm{a}_{3}^{3+}-\mathrm{O}^{2-}-\mathrm{Cu}^{2+}(4 \mathrm{b}) $$

  • The peroxide is very tightly bound (Kd = 10^-10)

Reaction 5

Formation of water $$ \mathrm{a}{3}^{3+}-\mathrm{O}^{2-}-\mathrm{Cu}^{2+}+2 \mathrm{c}^{2+}+2 \mathrm{H}^{+} \leftarrow \frac{\mathrm{K} 5=1 \times 10^{25}}{} \rightarrow 2 \mathrm{c}^{3+}+\mathrm{a}{3}^{3+}-\mathrm{Cu}^{2+}+2 \mathrm{H}_{2} \mathrm{O}(5) $$

Steady-state kinetic equations for the cytochrome c oxidase model.

  • The rates of the intermediate reactions (rxn 3 and rxn 5) have been shown to be fast compared with the net rate of oxygen reduction
  • The two-electron reduction of intermediates III and IV to V (reactions 4A and 4B) is irreversible, and therefore rate limiting
  • For near-equilibrium reactions, the energy loss is negligible and the energy conserved in the intermediate reactions is independent of the internal mechanism. This means energy coupling can be accurately represented by the appropriate energy barrier without specifying the mechanism

Cyt c reduction to turn number (TN)

  • The straight-line relationship predicted for Q (pmf?) less than 0.160 V is consistent with that observed experimentally for mitochondrial suspensions treated with uncoupler
Dependence of the rate of oxygen reduction on the fraction of cytochrome c reduced
The pH dependence of the relationship of cytochrome c turnover number to reduced fraction of cyt c

  • The high degree of control of the rate of oxygen reduction by cytochrome c oxidase is readily observed. At pH 7.35 and 25% reduction of cytochrome c, for example, the TN is about 1 s−1 for the coupled mitochondria, 12 s−1 for an ATP/ADP[Pi] of 300 M−1, and 60 s−1 for uncoupled mitochondria
The predicted dependence of the oxygen pressure for half-maximal respiratory rate as a function of the energy state (Q)

  • The oxygen pressure for a 50% decrease in respiratory rate (P50) is a function of the energy state (Q) decreasing from 20 torr when Q is 0.300 V to near 4 torr when Q is decreased to 0.200 V
  • This decrease in P50 (with decrease in Q) is accompanied by a large increase in respiratory rate (cytochrome c TN), and therefore is not comparable to experimental measurements of the P50 for respiration
Calculated changes in energy state, [ADP] and [AMP] for constant respiratory rate, and cytochrome c reduction as the oxygen in the medium decreases

  • During oxygen depletion the ADP and AMP concentrations increased by 4.9-fold and 24-fold, respectively, with no significant change in [ATP] and a 10% increase in [Pi]
The calculated levels of the intermediates in oxygen reduction as a function of the energy state (Q) as predicted by the steady-state rate expression for the model


  • During isolation of mitochondria, even from soft tissue such as liver, there is extensive damage to both structure and function

  • Both state 4 and state 3 (5) are experimental artifacts resulting from mitochondrial damage during isolation combined with nonphysiological assay conditions.

    • The state 4 respiration by suspensions of isolated mitochondria is due to isolation-induced leak(s) in energy coupling. The leak is positively related to the energy level and oxygen levels.
  • Muscles of insects, hummingbirds, and humans can reach metabolic rates from 50 to more than 100 times their resting rates, and ATP is efficiently produced at both conditions.

  • In well-coupled mitochondria (Q = 300 mV), nearly 100% of the oxidase can be in the form of the intermediate, V, consistent with spectral change (high-spin ferric form to the low-spin ferric form) in the experiments. In uncoupled mitochondria, the concentration is vanishingly small, and this would be expected for preparations of isolated cytochrome oxidase as well

  • The oxygen dependence of respiration described by the steady-state rate expression for the model is consistent with the behavior of isolated mitochondria and intact cells

  • The increases in free ADP and AMP, however, are large enough (Fig. 7) to cause substantial alterations in metabolism (e.g. AMPK pathway)

Appendix A

Steady-state kinetic expression for oxygen reduction by cytochrome c oxidase (Fig. 1).

Assumptions: Reactions 4A and 4B, is irreversible and the intermediates are in the steady state.


  1. Wilson DF, Vinogradov SA. Mitochondrial cytochrome c oxidase: mechanism of action and role in regulating oxidative phosphorylation. J. Appl. Physiol. 2014;117(12):1431-1439. doi:10.1152/japplphysiol.00737.2014. APS↩︎