Skip to main content
SpringerLink
Log in

XFEL Studies on Bovine Heart Cytochrome c Oxidase

  • Chapter
  • First Online:
Cytochrome Complexes: Evolution, Structures, Energy Transduction, and Signaling

Part of the book series: Advances in Photosynthesis and Respiration ((AIPH,volume 41))

  • 1635 Accesses

Summary

Cytochrome c oxidase (CcO) has four redox-active metal sites, Fe a , Fe a3 , CuA and CuB. Each site reversibly receives one electron equivalent. The four electron equivalents required to reduce the bound O2 are sequentially transferred from cytochrome c and each is coupled to the pumping of one proton equivalent. The purified enzyme fraction as prepared (the resting oxidized form) is not involved in catalytic turnover since one electron reduction of the resting oxidized form is not coupled to proton pumping. Redox and resonance Raman data suggest that a peroxide molecule bridges Fe a3 and CuB in the O2 reduction site. X-ray structural analyses show that the O-O bond length is 1.7 Å, which is significantly longer than that of the typical peroxide bridge between two metals (1.5 Å), suggesting an activated state of the bound peroxide. On the other hand, investigation of a bacterial CcO produced the proposal that the bound peroxide is not the intrinsic ligand; instead the peroxide is formed from hydroxyl radicals created by the strong X-ray radiation. Establishment of the ligand structure in the O2 reduction site is a prerequisite for elucidation of the proton-pumping mechanism.

The damage-free X-ray structure of CcO determed using the recently developed femtosecond pulse of an X-ray free-electron laser (XFEL) system shows the existence of a typical peroxide ligand with an O-O distance of 1.55 Å. Thus, the negatively-charged peroxide in the O2 reduction site blocks the proton-pump upon initial two electron reduction by suppressing the electron transfer from Fe a to Fe a3 (with Fe a oxidation driving the proton pump.).

This successful determination of a high resolution X-ray structure using femtosecond XFEL pulse validates the system for analysis of changes in X-ray structures over a physiologically-relevant time scale. This chapter includes a discussion for the new field, “Picobiology”, which is made possible by XFEL.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 259.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 329.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 329.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

A form:

The O2-bound form of CcO closely similar to oxymyoglobin, which appears in the catalytic turnover of CcO

A type CcO:

Group of CcOs containing three proton conducting pathways D, K, and H

B type CcO:

A group of CcOs containing only one proton conducting pathways corresponding to the K pathway of A type CcO

CcO:

Cytochrome c oxidase

CuA :

One of the copper sites of cytochrome c oxidase which is the initial electron acceptor from cytochrome c

CuB :

One of the copper sites of cytochrome c oxidase which forms the O2 reduction site with Fe a3

D-pathway:

One of the proton conducting pathways connecting N-phase and the O2 reduction site for transfer of protons for making water molecules

Fe a :

The low spin iron ion of one of the two heme As designated as heme a, contained in cytochrome c oxidase

Fe a3 :

The high spin iron ion of one of the two heme. As designated as heme a 3, contained in cytochrome c oxidase

fs:

Femtosecond (10−15 s)

H-pathway:

The proton conducting pathway connecting the N-phase with the P-phase for proton transfer and pumping

K-pathway:

One of the proton conducting pathways connecting the N-phase and the O2 reduction site for transfer of protons to make water molecules

N-phase:

Aqueous phase located in the inside of mitochondrial inner (or bacterial plasma) membrane

O form:

The fully oxidized form which appears under turnover conditions

P-phase:

Aqueous phase located in the outside of mitochondrial inner (or bacterial plasma) membrane

R form:

One of the intermediate species of the catalytic turnover of CcO in which both metals in the O2 reduction site are in the reduced state and free from any external ligand

SACLA:

The nick name of the XFEL facility constructed in Sayo Japan

XFEL:

X-ray free electron laser

References

  • Antonini E, Brunori M, Colosimo A, Greenwood C, Wilson MT (1977) Oxygen “pulsed” cytochrome c oxidase: functional properties and catalytic relevance. Proc Natl Acad Sci U S A 74:3128–3132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Aoyama H, Muramoto K, Shinzawa-Itoh K, Hirata K, Yamashita E, Tsukihara T, Ogura T, Yoshikawa S (2009) A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump. Proc Natl Acad Sci U S A 106:2165–2169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Artzatbanov VY, Konstantinov AA, Skulachev VP (1978) Involvement of intramitochondrial protons in redox reactions of cytochrome alpha. FEBS Lett 87:180–185

    Article  CAS  PubMed  Google Scholar 

  • Baker GM, Noguchi M, Palmer G (1987) The reaction of cytochrome oxidase with cyanide. Preparation of the rapidly reacting form and its conversion to the slowly reacting form. J Biol Chem 262:595–604

    CAS  PubMed  Google Scholar 

  • Bloch D, Belevich I, Jasaitis A, Ribacka C, Puustinen A, Verkhovsky MI, Wikström M (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci U S A 101:529–533

    Article  CAS  PubMed  Google Scholar 

  • Brandt U, Schägger H, von Jagow G (1989) Purification of cytochrome-c oxidase retaining its pulsed form. Eur J Biochem 182:705–711

    Article  CAS  PubMed  Google Scholar 

  • Burke JM, Kincaid JR, Peters S, Gagne RR, Collman JP, Spiro TG (1978) Structure-sensitive resonance Raman bands of tetraphenyl and “picket fence” porphyrin-iron complexes, including an oxyhemoglobin analog. J Am Chem Soc 100:6083–6088

    Article  CAS  Google Scholar 

  • Chang H-Y, Hemp J, Chen Y, Fee JA, Gennis RB (2009) The cytochrome ba 3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping. Proc Natl Acad Sci U S A 106:16169–16173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheesman MR, Oganesyan VS, Watmough NJ, Butler CS, Thomson AJ (2004) The nature of the exchange coupling between high-spin Fe(III) heme o 3 and CuBII in Escherichia coli quinol oxidase, cytochrome bo 3: MCD and EPR studies. J Am Chem Soc 126:4157–4166

    Article  CAS  PubMed  Google Scholar 

  • Collman JP, Sunderland CJ, Berg KE, Vance MA, Solomon EI (2003) Spectroscopic evidence for a heme-superoxide/Cu(I) intermediate in a functional model of cytochrome c oxidase. J Am Chem Soc 125:6648–6649

    Article  CAS  PubMed  Google Scholar 

  • Cooper CE, Salerno JC (1992) Characterization of a novel g’ = 2.95 EPR signal from the binuclear center of mitochondrial cytochrome c oxidase. J Biol Chem 267:280–285

    CAS  PubMed  Google Scholar 

  • Day EP, Peterson J, Sendova MS, Schoonover J, Palmer G (1993) Magnetization of fast and slow oxidized cytochrome c oxidase. Biochemistry 32:7855–7860

    Article  CAS  PubMed  Google Scholar 

  • Dodson ED, Zhao XJ, Caughey WS, Elliott CM (1996) Redox dependent interactions of the metal sites in carbon monoxide-bound cytochrome c oxidase monitored by infrared and UV/visible spectroelectrochemical methods. Biochemistry 35:444–452

    Article  CAS  PubMed  Google Scholar 

  • Du W-GH, Noodleman L (2013) Density functional study for the bridged dinuclear center based on a high-resolution X-ray crystal structure of ba 3 cytochrome c oxidase from Thermus thermophilus. Inorg Chem 52:14072–14088

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hirata K, Shinzawa-Itoh K, Yano N, Takemura S, Kato K, Hatanaka M, Muramoto K, …, Ago H (2014) Determination of damage-free crystal structure of an X-ray-sensitive protein using an XFEL. Nat Methods 11:734–736

    Google Scholar 

  • Isaacs NS (1995) Physical Organic Chemistry, 2nd edn. Longman Scientific & Technical, Harlow

    Google Scholar 

  • Kaila VRI, Verkhovsky MI, Wikström M (2010) Proton-coupled electron transfer in cytochrome oxidase. Chem Rev 110:7062–7081

    Article  CAS  PubMed  Google Scholar 

  • Kim E, Chufán EE, Kamaraj K, Karlin KD (2004) Synthetic models for heme-copper oxidases. Chem Rev 104:1077–1133

    Article  CAS  PubMed  Google Scholar 

  • Koepke J, Olkhova E, Angerer H, Müller H, Peng G, Michel H (2009) High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: new insights into the active site and the proton transfer pathways. Biochim Biophys Acta 1787:635–645

    Article  CAS  PubMed  Google Scholar 

  • Konstantinov AA, Siletsky S, Mitchell D, Kaulen A, Gennis RB (1997) The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer. Proc Natl Acad Sci U S A 94:9085–9090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kubo M, Nakashima S, Yamaguchi S, Ogura T, Mochizuki M, Kang J, Tateno M, …, Yoshikawa S (2013) Effective pumping proton collection facilitated by a copper site (CuB) of bovine heart cytochrome c oxidase, revealed by a newly developed time-resolved infrared system. J Biol Chem 288:30259–30269

    Google Scholar 

  • Mitchell R, Brown S, Mitchell P, Rich PR (1992) Rates of cyanide binding to the catalytic intermediates of mammalian cytochrome c oxidase, and the effects of cytochrome c and poly(L-lysine). Biochim Biophys Acta 1100:40–48

    Article  CAS  PubMed  Google Scholar 

  • Mochizuki M, Aoyama H, Shinzawa-Itoh K, Usui T, Tsukihara T, Yoshikawa S (1999) Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase. J Biol Chem 274:33403–33411

    Article  CAS  PubMed  Google Scholar 

  • Moody AJ (1996) ‘As prepared’ forms of fully oxidised haem/Cu terminal oxidases. Biochim Biophys Acta 1276:6–20

    Article  PubMed  Google Scholar 

  • Muramoto T, Ohta K, Shinzawa-Itoh K, Kanda K, Taniguchi M, Nabekura H, Yamashita E, …, Yoshikawa S (2010) Bovine cytochrome c oxidase structures enable O2 reduction with minimization of reactive oxygens and provide a proton-pumping gate. Proc Natl Acad Sci U S A 107:7740–7745

    Google Scholar 

  • Nakamoto K (1997) Infrared and Raman spectra of inorganic and coordination compounds, Part B, 5th edn. Wiley, New York

    Google Scholar 

  • Naqui A, Kumar C, Ching YC, Powers L, Chance B (1984) Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding. Biochemistry 23:6222–6227

    Article  CAS  PubMed  Google Scholar 

  • Ogura T, Takahashi S, Hirota S, Shinzawa-Itoh K, Yoshikawa S, Appelman EH, Kitagawa T (1993) Time-resolved resonance Raman elucidation of the pathway for dioxygen reduction by cytochrome c oxidase. J Am Chem Soc 115:8527–8536

    Article  CAS  Google Scholar 

  • Pereira MM, Sousa FL, Veríssimo AF, Teixeira M (2008) Looking for the minimum common denominator in haem-copper oxygen reductases: towards a unified catalytic mechanism. Biochim Biophys Acta 1777:929–934

    Article  CAS  PubMed  Google Scholar 

  • Proshlyakov DA, Ogura T, Shinzawa-Itoh K, Yoshikawa S, Appelman EH, Kitagawa T (1994) Selective resonance Raman observation of the “607 nm” form generated in the reaction of oxidized cytochrome c oxidase with hydrogen peroxide. J Biol Chem 269:29385–29388

    CAS  PubMed  Google Scholar 

  • Proshlyakov DA, Ogura T, Shinzawa-Itoh K, Yoshikawa S, Kitagawa T (1996) Microcirculating system for simultaneous determination of Raman and absorption spectra of enzymatic reaction intermediates and its application to the reaction of cytochrome c oxidase with hydrogen peroxide. Biochemistry 35:76–82

    Article  CAS  PubMed  Google Scholar 

  • Qin L, Hiser C, Mulichak A, Garavito RM, Ferguson-Miller S (2006) Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase. Proc Natl Acad Sci U S A 103:16117–16122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sakaguchi M, Shinzawa-Itoh K, Yoshikawa S, Ogura T (2010) A resonance Raman band assignable to the O-O stretching mode in the resting oxidized state of bovine heart cytochrome c oxidase. J Bioenerg Biomembr 42:241–243

    Article  CAS  PubMed  Google Scholar 

  • Siletsky SA, Konstantinov AA (2012) Cytochrome c oxidase: charge translocation coupled to single-electron partial steps of the catalytic cycle. Biochim Biophys Acta 1817:476–488

    Article  CAS  PubMed  Google Scholar 

  • Tiefenbrunn T, Liu W, Chen Y, Katritch V, Stout CD, Fee JA, Cherezov V (2011) High resolution structure of the ba 3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment. PLoS One 6:e22348

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsubaki M, Nagai K, Kitagawa T (1980) Resonance Raman spectra of myoglobins reconstituted with spirographis and isospirographis hemes and iron 2,4-diformylprotoporphyrin IX. Effect of formyl substitution at the heme periphery. Biochemistry 19:379–385

    Article  CAS  PubMed  Google Scholar 

  • Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R, …, Yoshikawa S (1995) Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A. Science 269:1069–1074

    Google Scholar 

  • Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R, …, Yoshikawa S (1996) The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 272:1136–1144

    Google Scholar 

  • van Wart HE, Zimmer J (1985) Resonance Raman evidence for the activation of dioxygen in horseradish oxyperoxidase. J Biol Chem 260:8372–8377

    PubMed  Google Scholar 

  • Verkhovsky MI, Jasaitis A, Verkhovskaya ML, Morgan JE, Wikström M (1999) Proton translocation by cytochrome c oxidase. Nature 400:480–483

    Article  CAS  PubMed  Google Scholar 

  • Verkhovsky MI, Tuukkanen A, Backgren C, Puustinen A, Wikström M (2001) Charge translocation coupled to electron injection into oxidized cytochrome c oxidase from Paracoccus denitrificans. Biochemistry 40:7077–7083

    Article  CAS  PubMed  Google Scholar 

  • Weng LC, Baker GM (1991) Reaction of hydrogen peroxide with the rapid form of resting cytochrome oxidase. Biochemistry 30:5727–5733

    Article  CAS  PubMed  Google Scholar 

  • Wikström M (2012) Active site intermediates in the reduction of O2 by cytochrome oxidase, and their derivatives. Biochim Biophys Acta 1817:468–475

    Article  PubMed  Google Scholar 

  • Wrigglesworth JM (1984) Formation and reduction of a ‘peroxy’ intermediate of cytochrome c oxidase by hydrogen peroxide. Biochem J 217:715–719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wrigglesworth JM, Elsden J, Chapman A, Van der Water N, Grahn MF (1988) Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB. Biochim Biophys Acta 936:452–464

    Article  CAS  PubMed  Google Scholar 

  • Yonetani T (1961) Studies on cytochrome oxidase III. Improved preparation and some properties. J Biol Chem 236:1680–1688

    CAS  PubMed  Google Scholar 

  • Yoshikawa S, Shinzawa-Itoh K, Nakashima R, Yaono R, Yamashita E, Inoue N, Yao M, …, Tsukihara T (1998) Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase. Science 280:1723–1729

    Google Scholar 

  • Yoshikawa S, Muramoto K, Shinzawa-Itoh K (2011) Proton-pumping mechanism of cytochrome c oxidase. Ann Rev Biophys 40:205–223

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work is supported by a Grant-in-Aid for the Global Center of Excellence Program, for the Targeted Protein Research Program, for Scientific Research (A) 2247012 and (B) 26234567, by the Japanese Ministry of Education, Culture, Sports, Science and Technology, and by JST, CREST. S. Yoshikawa is a Senior Visiting Scientist in the RIKEN Harima Institute.

Author information

Authors and Affiliations

  1. Picobiology Institute, Graduate School of Life Sciences, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo, 678-1297, Japan

    Shinya Yoshikawa

Authors
  1. Shinya Yoshikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shinya Yoshikawa .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA

    William A. Cramer

  2. Department of Biology, University of Wisconsin-Oshkosh, Oshkosh, Wisconsin, USA

    Toivo Kallas

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media Dordrecht

About this chapter

Cite this chapter

Yoshikawa, S. (2016). XFEL Studies on Bovine Heart Cytochrome c Oxidase. In: Cramer, W., Kallas, T. (eds) Cytochrome Complexes: Evolution, Structures, Energy Transduction, and Signaling. Advances in Photosynthesis and Respiration, vol 41. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-7481-9_18

Download citation

Publish with us

Policies and ethics

Search

Navigation