Electron transfer in monomeric forms of beef and shark heart cytochrome c oxidase

Abstract

Beef heart cytochrome c oxidase is dimeric in reconstituted membranes and in nonionic detergents at physiological pH [Henderson, R., Capaldi, R. A., & Leigh, J. (1977) J. Mol. Biol. 112, 631; Robinson, N.C., & Capaldi, R. A. (1977) Biochemistry 16, 375], raising the possibility that this aggregation state is a prerequisite for enzymatic activity. A procedure for dissociating the enzyme into monomers is presented. This involves treating the protein with high concentrations of Triton X-100 at pH 8.5. The electron transfer activity of the monomer is comparable to that of the dimer under identical assay conditions. The beef heart cytochrome c oxidase monomer was found to be heterogeneous in hydrodynamic studies, probably due to dissociation of associated polypeptides, including subunit III. Monomer molecular weights in the range 129 000-160 000 were obtained. Previous studies have indicated that shark heart cytochrome c oxidase is monomeric under physiological conditions. Sedimentation equilibrium studies reported here confirm this. The elasmobranch enzyme, with a similar polypeptide composition to that of beef enzyme, was determined to have a molecular weight of 158 000.

The site of the redox‐linked proton pump in eukaryotic cytochrome c oxidases

“…At pH 6.~7.5 (25°C) we find the turnover number of these preparations to be 460470 s -I (mol ferrocytochrome c oxidized/mol enzyme). This is significantly higher than the maximum value previously determined for Yonetani type preparations of shark oxidases [4,13]. At pH 9.0 there is no measurable activity under otherwise standard conditions.…”

“…Fully oxidized derivatives of Hartzell-Beinert type preparations of both beef and shark cytochrome c oxidase show a marked sensitivity in the position of the Soret band to pH. Unlike the beef enzyme, shark oxidase does not undergo pH-dependent changes in aggregation state [13] and so, the observed spectral shifts cannot be attributed to factors linked to association-dissociation processes. Somewhat surprisingly, the Yonetani type enzyme preparations showed very little evidence of pH-dependent spectral behaviour.…”

Spectroscopic analysis of the cytochrome c oxidase-cytochrome c complex: circular dichroism and magnetic circular dichroism measurements reveal change of cytochrome c heme geometry imposed by complex formation.

“…This buffer was used for all spectroscopic measurements. The enzyme was brought into this buffer by ion-exchange chromatography (16). Concentrations of heme c and heme aa3 were determined as before (11 for the oxidase-ferricytochrome c complex ( ), equivalent to the difference between the spectra shown in A; difference CD spectrum of ferricytochrome c measured at pH 11 and pH 7.4 ( ).…”

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

“…In the present study, it has been discovered that the kinetics of compound F reduction are strongly influenced by treatments of the enzyme that traditionally have been used to convert the bovine enzyme to the monomeric form. 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.…”

“…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.…”