A comparison of three preparations of cytochrome c oxidase. Optical absorbance spectra, EPR spectra and reaction towards ligands.
Three preparations of cytochrome c oxidase, the preparation as traditionally prepared in our laboratory as described by Van Buuren (1992; PhD Thesis, University of Amsterdam), a preparation according to Volpe and Caughey (Biochem. Biophys. Res. Commun. 61 (1974) 502-509) and a preparation of 'fast' cytochrome c oxidase (Brandt, U., Schägger, H. and Von Jagow, G. (1989) Eur. J. Biochem. 182, 705-711), are compared in their reaction with cyanide and carbon monoxide. The reaction with cyanide is nearly as fast for the Van Buuren preparation as for the 'fast' preparation, but much slower for the Volpe-Caughey preparation. Mixed-valence cytochrome c oxidase (cytochrome a3 and CuB reduced with carbon monoxide bound and cytochrome a and CuA oxidized) is prepared by anaerobic incubation with carbon monoxide. With the Van Buuren preparation complete formation of the species takes 4 h, whereas with the Volpe-Caughey preparation it takes 20 h. Longer incubation under CO results in partial reduction of cytochrome a and CuA. With the 'fast' preparation mixed-valence cytochrome c oxidase is formed after more than 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 towards cyanide and towards carbon monoxide in cytochrome c oxidase of all three preparations. Furthermore, optical and EPR spectra of the preparations of cytochrome c oxidase are compared. The Volpe-Caughey preparation has an intense g' = 12 EPR-signal, the Van Buuren preparation has 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 preparations in the Soret region are similar with a maximum at 424 nm. Only the 'fast' preparation as isolated was completely oxidized, whereas the other preparations were partially reduced. It was concluded that differences in the reaction of cytochrome c oxidase with ligands are determined by the internal or external ligand bound to the cytochrome a3-CuB couple.
E.C. Slater Institute, University of Amsterdam, The Netherlands.
A L
AL
Lodder
B F
BF
van Gelder
D002248:Carbon Monoxide / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism; D017936:Cytochrome a Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005561:Formates / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D003486:Cyanides; D017936:Cytochrome a Group; D005561:Formates; C030544:formic acid; D003300:Copper; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV