A COMPARISON OF 3 PREPARATIONS OF CYTOCHROME-C-OXIDASE - OPTICAL ABSORBENCY SPECTRA, EPR-SPECTRA AND REACTION TOWARDS LIGANDS

Three preparations of cytochrome c oxidase, the preparation as traditi onally prepared in our laboratory as described by Van Buuren (1992; Ph D Thesis, University of Amsterdam), a preparation according to Volpe a nd Caughey (Biochem. Biophys. Res. Commun. 61 (1974) 502-509) and a pr eparation of 'fast' cytochrome c oxidase (Brandt, U., Schagger, H. and Von Jagow, G. (1989) fur. J. Biochem. 182, 705-711), are compared in their reaction with cyanide and carbon monoxide. The reaction with cya nide is nearly as fast for the Van Buuren preparation as for the 'fast ' preparation, but much slower for the Volpe-Caughey preparation. Mixe d-valence cytochrome c oxidase (cytochrome a(3) and Cu-B reduced with carbon monoxide bound and cytochrome a and Cu-A oxidized) is prepared by anaerobic incubation with carbon monoxide. With the Van Buuren prep aration complete formation of the species takes 4 h, whereas with the Volpe-Caughey preparation it takes 20 h. Longer incubation under CO re sults in partial reduction of cytochrome a and Cu-A. With the 'fast' p reparation mixed-valence cytochrome c oxidase is formed after more tha n one day of incubation with CO, but it is stable for at least 3 days. The presence of oxidized cytochrome c did enhance the reactivity towa rds cyanide and towards carbon monoxide in cytochrome c oxidase of all three preparations. Furthermore, optical and EPR spectra of the prepa rations of cytochrome c oxidase are compared. The Volpe-Caughey prepar ation has an intense g' = 12 EPR-signal, the Van Buuren preparation ha s hardly any g' = 12 signal and the 'fast' preparation has no g' = 12 signal. In the 'fast' preparation the low-spin heme signal is shifted (from g = 3.00 to g = 2.97). The absorbance spectra of the three prepa rations in the Soret region are similar with a maximum at 424 nm. Only the 'fast' preparation as isolated was completely oxidized, whereas t he other preparations were partially reduced. It was concluded that di fferences in the reaction of cytochrome c oxidase with ligands are det ermined by the internal or external ligand bound to the cytochrome a(3 )-Cu-B couple.