Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans

Tuomas Haltia, Nora Semo, Ernesto I Freire, Felix M. Goñi, Ernesto Freire

Research output: Contribution to journalArticle

Abstract

The structural stability of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans has been measured by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Contrary to the mammalian enzyme or the yeast enzyme, which are composed of 13 subunits, the bacterial enzyme has only three or four subunits, thus providing a unique opportunity to examine the magnitude of the forces that stabilize this enzyme and to establish accurate structural assignments of events observed calorimetrically. In this paper, experiments have been performed with the wild-type enzyme and with a mutant enzyme lacking subunit III. Our results show that subunits I and II form a highly cooperative complex which denatures as a single cooperative unit at 67°C, while subunit III is less stable and denatures 20°C earlier. Reduction of the enzyme causes a large increase in the stability of subunits I and II but has absolutely no effect on subunit III. Despite the lack of a strong interaction between subunit III and the catalytic subunits, the absence of subunit III leads to a turnover-induced loss of electron-transfer activity. The magnitude of the energetic parameters and the infrared spectroscopic experiments indicate that the enzyme does not completely unfold upon thermal denaturation and that significant degrees of structure are preserved. The amount of native α-helix structure, which is 45% in the native state, decreases only to 30% after thermal denaturation. Presumably, the residual helical structure existing after thermal denaturation belongs to the intramembranous portions of the protein. The calorimetric behavior of subunit III does not fully conform to that expected for a highly α-helical membrane protein. The picture that emerges from these experiments is that, in the temperature-denatured form of the enzyme, most of the extramembranous structural elements are denatured while most of the intramembranous secondary structure is maintained even though native tertiary interactions appear to be disrupted.

Original languageEnglish (US)
Pages (from-to)9731-9740
Number of pages10
JournalBiochemistry®
Volume33
Issue number32
StatePublished - 1994

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Paracoccus denitrificans
Electron Transport Complex IV
Thermodynamics
Enzymes
Denaturation
Hot Temperature
Membrane Proteins
Experiments
Differential Scanning Calorimetry
Fourier Transform Infrared Spectroscopy
Yeast
Differential scanning calorimetry
Catalytic Domain
Yeasts
Electrons
Infrared radiation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans. / Haltia, Tuomas; Semo, Nora; Freire, Ernesto I; Goñi, Felix M.; Freire, Ernesto.

In: Biochemistry®, Vol. 33, No. 32, 1994, p. 9731-9740.

Research output: Contribution to journalArticle

Haltia, T, Semo, N, Freire, EI, Goñi, FM & Freire, E 1994, 'Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans', Biochemistry®, vol. 33, no. 32, pp. 9731-9740.
Haltia, Tuomas ; Semo, Nora ; Freire, Ernesto I ; Goñi, Felix M. ; Freire, Ernesto. / Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans. In: Biochemistry®. 1994 ; Vol. 33, No. 32. pp. 9731-9740.
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