Studies of the kinetics of oxidation of cytochrome c by cytochrome c oxidase: Comparison of the reactions of purified and membrane-bound oxidase

Abstract

Purified cytochrome oxidase was compared to the oxidase on submitochondrial particles with respect to reactions with added soluble cytochrome c. The reactions studied were the spectrophotometric assay of the oxidation of ferrocytochrome c, and the polarographic measurement of the rate of O 2 uptake in the presence of N,N,N′,N′-tetramethylphenyl-enediamine plus ascorbate and cytochrome c. The oxidase activity of the preparations expressed in terms of their concentration of cytochrome aa 3 was lower with the purified preparation than with the membrane-bound preparation. Apparent K M values for low concentrations of cytochrome c (0.05 to 0.25 μ m) derived from plots of v S versus v are similar for the two types of preparation when obtained either by the spectrophotometric or polarographic assays. The apparent K M values for higher concentrations of cytochrome c (0.5 to 5.0 μM) are similar in the spectrophotometric assays but different in the polarographic determinations. At pH 7.8 in buffers with nonbinding ions, the rates of O 2 uptake measured polarographically were greater than the rates of O 2 uptake calculated from the spectrophotometric measurements of the rates of oxidation of ferrocytochrome c ( L. Smith, H. C. Davies, and M. E., Nava (1979) Biochemistry 18, 3140–3146 ). The O 2 uptake observed in the polarographic method in excess of that corresponding to the oxidation of ferrocytochrome c is much greater with the membrane-bound (30-fold) than with the purified oxidase (three- to four-fold difference). This implies a decreased ability of the purified preparation to form a complex with a high turnover rate which can be repeatedly reduced by the N,N,N′,N′-tetramethylphenylenediamine plus ascorbate. The extent of cytochrome c which remains oxidized during the aerobic steady state in the presence of the reducing agents is less for the purified than for the particulate preparation, suggesting that the reactive complex of cytochrome c and cytochrome oxidase is not as readily formed with the purified preparation as with the SMP.