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Kinetic studies on cytochrome c oxidase inserted into liposomal vesicles. Effect of ionophores.

ABSTRACT: Cytochrome c oxidase from ox heart was inserted into artificial liposomal vesicles obtained by sonication of purified soya-bean phospholipids. The cytochrome oxidase vesicles showed a respiratory control ratio of about 2. Spectroscopic properties in the visible and Soret regions and kinetics of CO binding are similar to those of the soluble oxidase. The catalytic efficiency of the cytochrome oxidase vesicles in oxidizing cytochrome c increases as a result of the formation of the 'pulsed' form of the oxidase and of the presence in the reaction mixture of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and nonactin. Analysis of the experimental results obtained under several conditions supports the conclusions that: (i) the alkalinization of the internal microenvironment in the liposomal vesicle is not by itself responsible for the decrease in catalytic activity; (ii) the electrical potential difference created during turnover by proton consumption and/or pumping through the liposome wall is an important mechanism of control in the chain of events leading to the oxidation of external cytochrome c.

SUBMITTER: Sarti P

PROVIDER: S-EPMC1154058 | biostudies-other | 1983 Jan

REPOSITORIES: biostudies-other

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Project description:Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa? CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O? reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O? reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P?F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F?O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P?F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F?O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.

Project description:Thermally induced transitions of the 13-subunit integral membrane protein bovine cytochrome c oxidase (CcO) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). Thermal denaturation of dodecyl maltoside solubilized CcO proceeds in two consecutive, irreversible, kinetically driven steps with the apparent transition temperatures at ∼ 51°C and ∼ 61°C (5μM CcO at scan rate of 1.5 K/min). The thermal denaturation data were analyzed according to the Lyubarev and Kurganov model of two consecutive irreversible steps. However, because of the limitation of the model to describe the complex mechanism of the thermal denaturation of CcO, the obtained results were utilized only for comparison purposes of kinetic stabilities of CcO under specific protein concentration (5μM) and scan rate (1.5 K/min). This enabled us to show that both the amphiphilic environment and the self-association state of CcO affect its kinetic stability. Kinetic stabilities of both steps are significantly decreased when all of the phospholipids are removed from CcO by phospholipase A2 (the half-life decreases at 37°C). Conversely, dimerization of CcO induced by sodium cholate significantly increases its kinetic stability of only the first step (the half-life increases at 37°C). Protein concentration-dependent nonspecific oligomerization also indicate mild stabilization of CcO. Both, reversed-phase high-performance liquid chromatography (HPLC) and SDS-PAGE subunit analysis reveal that the first step of thermal denaturation involves dissociation of subunits III, VIa, VIb, and VIIa, whereas the second step is less well defined and most likely involves global unfold and aggregation of the remaining subunits. Electron transport activity of CcO decreases in a sigmoidal manner during the first transition and this dependence is very well described by kinetic parameters for the first step of the thermal transition. Therefore, dissociation of subunit III and/or VIIa is responsible for temperature-induced inactivation of CcO because VIa and VIb can be removed from CcO without affecting the enzyme activity. These results demonstrate an important role of tightly bound phospholipids and oligomeric state (particularly the dimeric form) of CcO for kinetic stability of the protein.

Project description:In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.

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Kinetic studies on cytochrome c oxidase inserted into liposomal vesicles. Effect of ionophores.

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