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A mechanistic principle for proton pumping by cytochrome c oxidase


In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c1,2,3,4,5,6,7. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane8 per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.

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Figure 1: Structural and functional properties of cytochrome c oxidase.
Figure 2: Absorbance changes of the dye phenol red associated with proton release and uptake on reaction of the four-electron reduced Cyt c O with O2.
Figure 3: A schematic mechanism that accounts for proton pumping without simultaneous electron transfer.


  1. Gennis, R. B. Coupled proton and electron transfer reactions in cytochrome oxidase. Front. Biosci. 9, 581–591 (2004)

    CAS  Article  Google Scholar 

  2. Wikström, M. Cytochrome c oxidase: 25 years of the elusive proton pump. Biochim. Biophys. Acta 1655, 241–247 (2004)

    Article  Google Scholar 

  3. Mills, D. A. & Ferguson-Miller, S. Understanding the mechanism of proton movement linked to oxygen reduction in cytochrome c oxidase: lessons from other proteins. FEBS Lett. 545, 47–51 (2003)

    CAS  Article  Google Scholar 

  4. Michel, H., Behr, J., Harrenga, A. & Kannt, A. Cytochrome c oxidase: structure and spectroscopy. Annu. Rev. Biophys. Biomol. Struct. 27, 329–356 (1998)

    CAS  Article  Google Scholar 

  5. Rich, P. R. The molecular machinery of Keilin's respiratory chain. Biochem. Soc. Trans. 31, 1095–1105 (2003)

    CAS  Article  Google Scholar 

  6. Brzezinski, P. Redox-driven membrane-bound proton pumps. Trends Biochem. Sci. 29, 380–387 (2004)

    CAS  Article  Google Scholar 

  7. Brunori, M., Giuffré, A. & Sarti, P. Cytochrome c oxidase, ligands and electrons. J. Inorg. Biochem. 99, 324–336 (2005)

    CAS  Article  Google Scholar 

  8. Wikström, M. K. F. Proton pump coupled to cytochrome c oxidase in mitochondria. Nature 266, 271–273 (1977)

    ADS  Article  Google Scholar 

  9. Babcock, G. T. How oxygen is activated and reduced in respiration. Proc. Natl Acad. Sci. USA 96, 12971–12973 (1999)

    ADS  CAS  Article  Google Scholar 

  10. Lanyi, J. K. & Luecke, H. Bacteriorhodopsin. Curr. Opin. Struct. Biol. 11, 415–419 (2001)

    CAS  Article  Google Scholar 

  11. Paula, S., Sucheta, A., Szundi, I. & Einarsdóttir, Ó. Proton and electron transfer during the reduction of molecular oxygen by fully reduced cytochrome c oxidase: a flow-flash investigation using optical multichannel detection. Biochemistry 38, 3025–3033 (1999)

    CAS  Article  Google Scholar 

  12. Ädelroth, P., Ek, M. & Brzezinski, P. Factors determining electron-transfer rates in cytochrome c oxidase: investigation of the oxygen reaction in the R. sphaeroides and bovine enzymes. Biochim. Biophys. Acta 1367, 107–117 (1998)

    Article  Google Scholar 

  13. Oliveberg, M., Hallén, S. & Nilsson, T. Uptake and release of protons during the reaction between cytochrome c oxidase and molecular oxygen: a flow-flash investigation. Biochemistry 30, 436–440 (1991)

    CAS  Article  Google Scholar 

  14. Zaslavsky, D., Kaulen, A. D., Smirnova, I. A., Vygodina, T. & Konstantinov, A. A. Flash-induced membrane potential generation by cytochrome c oxidase. FEBS Lett. 336, 389–393 (1993)

    CAS  Article  Google Scholar 

  15. Verkhovsky, M. I., Morgan, J. E., Verkhovskaya, M. L. & Wikström, M. Translocation of electrical charge during a single turnover of cytochrome c oxidase. Biochim. Biophys. Acta 1318, 6–10 (1997)

    CAS  Article  Google Scholar 

  16. Zhen, Y. J., Schmidt, B., Kang, U. G., Antholine, W. & Ferguson-Miller, S. Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: I. Spectral and functional properties. Biochemistry 41, 2288–2297 (2002)

    CAS  Article  Google Scholar 

  17. Karpefors, M., Ädelroth, P., Zhen, Y., Ferguson-Miller, S. & Brzezinski, P. Proton uptake controls electron transfer in cytochrome c oxidase. Proc. Natl Acad. Sci. USA 95, 13606–13611 (1998)

    ADS  CAS  Article  Google Scholar 

  18. Papa, S., Capitanio, N. & Villani, G. A cooperative model for protonmotive heme-copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase. FEBS Lett. 439, 1–8 (1998)

    CAS  Article  Google Scholar 

  19. Michel, H. The mechanism of proton pumping by cytochrome c oxidase. Proc. Natl Acad. Sci. USA 95, 12819–12824 (1998)

    ADS  CAS  Article  Google Scholar 

  20. Rich, P. R., Jünemann, S. & Meunier, B. Protonmotive mechanism of heme-copper oxidases. J. Bioenerg. Biomembr. 30, 131–138 (1998)

    CAS  Article  Google Scholar 

  21. Tsukihara, T. et al. The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process. Proc. Natl Acad. Sci. USA 100, 15304–15309 (2003)

    ADS  CAS  Article  Google Scholar 

  22. Wikström, M., Verkhovsky, M. I. & Hummer, G. Water-gated mechanism of proton translocation by cytochrome c oxidase. Biochim. Biophys. Acta 1604, 61–65 (2003)

    Article  Google Scholar 

  23. Namslauer, A., Pawate, A. S., Gennis, R. B. & Brzezinski, P. Redox-coupled proton translocation in biological systems: Proton shuttling in cytochrome c oxidase. Proc. Natl Acad. Sci. USA 100, 15543–15547 (2003)

    ADS  CAS  Article  Google Scholar 

  24. Svensson-Ek, M. et al. The X-ray crystal structures of wild-type and EQ(I-286) mutant cytochrome c oxidases from Rhodobacter sphaeroides. J. Mol. Biol. 321, 329–339 (2002)

    CAS  Article  Google Scholar 

  25. Brzezinski, P. & Larsson, G. Redox-driven proton pumping by heme-copper oxidases. Biochim. Biophys. Acta 1605, 1–13 (2003)

    CAS  Article  Google Scholar 

  26. Siegbahn, P. E. M., Blomberg, M. R. A. & Blomberg, M. L. Theoretical study of the energetics of proton pumping and oxygen reduction in cytochrome oxidase. J. Phys. Chem. B 107, 10946–10955 (2003)

    CAS  Article  Google Scholar 

  27. Popovic, D. M. & Stuchebrukhov, A. A. Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating. FEBS Lett. 566, 126–130 (2004)

    CAS  Article  Google Scholar 

  28. Brändén, M. et al. On the role of the K-proton transfer pathway in cytochrome c oxidase. Proc. Natl Acad. Sci. USA 98, 5013–5018 (2001)

    ADS  Article  Google Scholar 

  29. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)

    CAS  Article  Google Scholar 

  30. Heller, H., Schaefer, M. & Schulten, K. Molecular-dynamics simulation of a bilayer of 200 lipids in the gel and in the liquid-crystal phases. J. Phys. Chem. 97, 8343–8360 (1993)

    CAS  Article  Google Scholar 

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We would like to thank S. Ferguson-Miller for providing the M263L mutant strain of R. sphaeroides and I. Holmgren for invaluable help with preparation of CytcO and the SUVs.

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Correspondence to Peter Brzezinski.

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Faxén, K., Gilderson, G., Ädelroth, P. et al. A mechanistic principle for proton pumping by cytochrome c oxidase. Nature 437, 286–289 (2005).

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