Reaction of thionitrobenzoate‐modified yeast cytochrome <i>c</i> with monomeric and dimeric forms of beef heart cytochrome <i>c</i> oxidase

Darley‐Usmar, Victor; Georgevich, Gradimir; Capaldi, Roderick A.

doi:10.1016/0014-5793(84)80058-7

Cited by 20 publications

(2 citation statements)

References 22 publications

(17 reference statements)

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“…Functional studies on cytochrome c oxidase are often interpreted in terms of a dimeric model for the complex, i.e., two multisubunit complexes each containing two a hemes and two coppers. For example, inhibition of the electron-transport activity with arylazidocytochrome c derivatives indicates "half of site" reactivity, suggesting that the complex exists as a dimer during the enzymatic assay (Bisson et al, 1980); the highaffinity cytochrome c binding site has been postulated to be at the interface between the monomers within the dimeric complex (Darley-Usmar et al, 1984).…”

Section: Discussionmentioning

confidence: 99%

Triton-X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Robinson¹,

Talbert²

1986

Biochemistry

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Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Triton-X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Robinson¹,

Talbert²

1986

Biochemistry

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“…In contrast to the regulatory site mechanism, the nonlinearity is now the exclusive property of dimeric and oligomeric oxidase. The model is based on the topographical features of beef heart oxidase obtained by electron microscopy and image reconstruction (Fuller et al, 1979) and on the results from chemical cross-linking of cytochrome c to oxidase (Darley-Usmar et al, 1983).…”

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confidence: 99%

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

Michel¹,

Bosshard²

1989

Biochemistry

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The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)

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Evidence for dimer structure of proton-pumping cytochrome c oxidase, an analysis by radiation inactivation.

Sone¹,

Kosako²

1986

The EMBO Journal

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Cytochrome c oxidases, purified from bovine heart and the thermophilic bacterium PS3, were irradiated with a high‐energy electron beam. The proton transport activities of both preparations and their electron transfer activities decreased as single exponential functions of the radiation dosage. Applying the target theory with alkaline phosphatase as an internal standard, the following functional molecular weights were obtained for cytochrome c oxidation and H+ pumping: 63‐73 kd and 160‐220 kd, respectively, for the bovine enzyme, and 80‐100 kd and 190‐230 kd for the PS3 enzyme. The results suggest that a dimer structure is necessary for H+ pumping, while a core part of monomer (presumably the largest two subunits, i.e. subunits I and II) is sufficient for cytochrome c oxidation.

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Reaction of thionitrobenzoate‐modified yeast cytochrome c with monomeric and dimeric forms of beef heart cytochrome c oxidase

Cited by 20 publications

References 22 publications

Triton-X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Triton-X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

Evidence for dimer structure of proton-pumping cytochrome c oxidase, an analysis by radiation inactivation.

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