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

Abstract

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)

Exposure of Bovine Cytochrome c Oxidase to High Triton X-100 or to Alkaline Conditions Causes a Dramatic Change in the Rate of Reduction of Compound F

“…The apparent sedimentation coefficient of untreated bovine CcO in 5 mM Tris-Cl (pH 8.0) containing 0.1% lauryl maltoside was 11.7 S. The sedimentation coefficient of the CcO preparation incubated for 2 h in 0.1% lauryl maltoside at pH 10.0 in 5 mM Tris-Cl was 9.5 S, whereas that of the preparation treated with 5% Triton X-100 was 9.0 S. The sedimentation coefficient of 11.7 S for the untreated CcO is consistent with previous studies of the dimeric protein (10). Robinson et al (11) measured a sedimentation coefficient of 8.7 S for monomeric CcO treated with Triton X-100, which is consistent with the value of 9.0 S obtained in the present study.…”

“…The reduction of compound F occurs in a single slow phase with a rate constant of 450 s Ϫ1 for the bovine enzyme in 0.1% Triton X-100, which has been shown to consist of dimers and higher aggregates (11). Raising the Triton X-100 concentration to 1% resulted in biphasic kinetics with equal amplitudes for the fast and slow phases.…”

“…Electron Transfer between Heme a and Oxyferryl Heme a 3 in Bovine Cytochrome c Oxidase in Triton X-100 -The kinetics were further investigated in different concentrations of Triton X-100, which has also been shown to affect the quaternary state of bovine CcO (10,11). Electron transfer from heme a to oxyferryl heme a 3 was monophasic for bovine CcO equilibrated with 0.1% Triton X-100, with a rate constant of 450 s Ϫ1 at pH 8.0 (Table I).…”

“…Two different methods were used to prepare monomeric bovine cytochrome oxidase, incubation with Triton X-100 at pH 8.0 (11) or pH 8.5 (10), and incubation with lauryl maltoside at pH 10.0 (15). When dimeric bovine cytochrome oxidase is incubated with Triton X-100 (10,11), a fast phase of compound F reduction with a rate constant of 2400 s Ϫ1 becomes dominant, and the slow phase is nearly eliminated. Furthermore, the rate constant of the fast phase is independent of pH from pH 8.0 to 10.0.…”

Monomerization of cytochrome oxidase may be essential for the removal of subunit III

“…3. The enzyme was incubated at different detergent/protein ratios since the degree of monomerization was shown to increase with increasing Triton/protein ratio [15]. The oligomeric state after the incubations was assayed in parallel samples.…”