Purification of cytochrome‐<i>c</i> oxidase retaining its pulsed form

Brandt, Ulrich; Schägger, Hermann; Jagow, Gebhard von

doi:10.1111/j.1432-1033.1989.tb14882.x

Cited by 61 publications

(39 citation statements)

References 24 publications

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“…This type of enzyme with slow and multiphasic cyanide kinetics has been used for several decades (resting enzyme) and is now generally considered as artifactual. Its main characteristics are a very slow internal transfer of electrons from heme a to the heme a,/Cu, center and the presence of a g = 12 EPR-signal (Baker et al, 1987;Brandt et al, 1989;Moody et al, 1992). Due to this slow transfer (1 s-' as determined in Triton X-100, Mahapatro and Robinson, 1990), equilibrium is seldom achieved within the equilibration time of 5 min, a very slow decrease in the difference absorbance thus reflects the slow transfer of electrons from heme a to heme a,.…”

Section: Resultsmentioning

confidence: 99%

“…3 shows reductive and reoxidative titration curves of preparation 2. This enzyme is characterized by fast and monophasic cyanide kinetics, a rapid internal electron transfer and the absence of the g = 12 signal and is therefore termed fast enzyme (Baker et a]., 1987; Brandt et al, 1989;Moody et al, 1992). As in the discontinuous and continuous titrations of preparation 1 complete reduction is achieved after the addition of 4.3 mol electrodmol cytochrome-c oxidase.…”

Section: Resultsmentioning

confidence: 99%

“…This was obtained from Brandt et al (1989). The enzyme was after thawing solubilized in 0.05% dodecyl pa-maltoside (Calbiochem); 10 mM TrisHCl, pH 7.6.…”

Section: Preparationmentioning

confidence: 99%

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Stoichiometry and redox behaviour of metals in cytochrome‐c oxidase

Steffens

Soulimane

Wolff

et al. 1993

European Journal of Biochemistry

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The early observation of extra copper in preparations of cytochrome-c oxidase has recently lead to a renewed interest in its stoichiometry and possible redox function. In various, pure preparations (heme A contents close to the theoretical value of 9.79 nmoVmg protein for the 13-subunit bovine enzyme) protein-related metal stoichiometries of 3 Cu, 2 Fe, 1 Zn, 1 Mg/monomer with M, 204266 were determined.Despite the presence of five potential redox metal ions, reductive and reoxidative titrations indicate the presence of only four one-electron-accepting/donating species in the ligand-free enzyme.Participation of two copper ions in a binuclear copper site acting as a one-electron acceptor may explain both the observed copper stoichiometry and the redox behaviour. The homology of the Cterminal sequence of subunit I1 with one of the copper-binding sites in nitrous-oxide reductases provides possible ligands for complexing two copper ions in a binuclear center.Cytochrome-c oxidase is a multisubunit protein localized in the inner mitochondria1 membrane of eucaryotes and the plasma membrane of many aerobic bacteria. The solubilized enzyme transfers electrons from cytochrome-c (Cyt-c) to molecular oxygen, which is thereby reduced to form water. According to the reaction equation 4Cyt-c2+ + 8H+ + 0, = 4Cyt c3+ + 4H: + 2H20 the free energy of the in-vivo-catalyzed reaction is conserved as an electrochemical proton potential by the twofold process of water formation and vectorial pumping across the membrane (H:), depleting protons (H,+) from the inner compartment. Ever since the fist description of the enzyme, experiments have been undertaken aiming at an understanding of the reaction mechanism of this important enzyme. However, nobody has yet achieved a preparation of this complex membrane protein which fulfills all criteria of purity, stoichiometry, activity and monodispersity. In addition to the doubt which this situation imposes on investigation of its reaction mechanism, this may be the reason why analysis of its threedimensional structure, either by two-dimensional or threedimensional crystallization, has so far remained inconsistent or unsuccessful.Correspondence to G. Buse, Institut fir Biochemie, RheinischWestfdische Technische Hochschule Aachen, Klinikum Pauwelsstrasse 30, W-5100 Aachen, Germany Fax: +49 241 80 88851. Abbreviations. ICP-AES, inductively coupled plasma atomicemission spectroscopy ; Pth, phenylthiohydantoin; EXAFS, extended X-ray absorption fine structure; T,max, maximal turnover number.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Stoichiometry and redox behaviour of metals in cytochrome‐c oxidase

Steffens

Soulimane

Wolff

et al. 1993

European Journal of Biochemistry

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“…Fortunately, there is no dearth of useful enzyme preparation procedures published. [20][21][22][23] Although involving some initial work and expense, any extensive projects along these lines would benefit from a stable, reliable, in-house source of CCO in quantity.…”

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confidence: 99%

Near-Infrared Irradiation Photobiomodulation: The Need for Basic Science

Quirk¹,

Whelan²

2011

Photomedicine and Laser Surgery

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“…Cytochrome c oxidase was prepared from bovine hearts using the method described by Brandt et al (22). The enzyme was solubilized from mitochondrial membranes using Triton X-100, followed by hydroxyapatite chromatography and exchange of Triton X-100 for 1% (wt/vol) dodecyl D-maltoside (see ref.…”

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confidence: 99%

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Ädelroth

Sigurdson²,

Hallén³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron-and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from -3 ,us to several milliseconds, reflecting internal electrontransfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV =

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Purification of cytochrome‐c oxidase retaining its pulsed form

Cited by 61 publications

References 24 publications

Stoichiometry and redox behaviour of metals in cytochrome‐c oxidase

Stoichiometry and redox behaviour of metals in cytochrome‐c oxidase

Near-Infrared Irradiation Photobiomodulation: The Need for Basic Science

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

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