Influence of non-esterified fatty acids on respiratory control of reconstituted cytochrome-c oxidase.

Bovine heart cytochrome-c oxidase was reconstituted in liposomes (asolectin) and the activity measured in the presence and absence of uncoupler at increasing concentrations of non-esterified fatty acids. Palmitic and stearic acids resulted in a decrease of about 40% in the respiratory control ratio at a concentration of 1 microM, when measured using a spectrophotometric procedure but not with a polarographic assay method. At higher fatty acid concentrations no further change was found. A 50% decrease in respiratory control was determined when the enzyme was reconstituted in pure phosphatidylcholine containing 2% cardiolipin. The respiratory control of reconstituted cytochrome-c oxidase from bovine liver was not influenced by fatty acids.

Regulation of the H+/e- stoichiometry of cytochrome c oxidase from bovine heart by intramitochondrial ATP/ADP ratios.

This paper describes the effect of intramitochondrial ATP/ADP ratios on the H+/e- stoichiometry of reconstituted cytochrome c oxidase (COX) from bovine heart. At 100% intraliposomal ATP the H+/e- stoichiometry of the reconstituted enzyme is decreased to half of the value measured below 98% intraliposomal ATP (above 2% ADP), while it remains constant up to 100% ADP. The decrease is obtained with different COX preparations, independent of the absolute value of the H+/e- stoichiometry. Decrease of H+/e- stoichiometry is prevented by preincubation of the enzyme with a tissue-specific monoclonal antibody to subunit VIa-H (heart type). Tissue-specific regulation of the efficiency of energy transduction in COX of muscle mitochondria could have a physiological function in maintaining the body temperature at rest or sleep, i.e. at low ATP expenditure.

Specific and reversible activation and inactivation of the mitochondrial phosphate carrier by cardiolipin and nonionic detergents, respectively.

The phosphate carrier of pig heart mitochondria was solubilized with Triton X-100 and purified by chromatography on hydroxylapatite. Incubation of the phosphate carrier fraction with cardiolipin stimulated the reconstituted [32P]phosphate exchange activity in liposomes, whereas increased Triton X-100 concentrations inhibited it. The effects of cardiolipin and Triton X-100 are reversible. The activation by cardiolipin is highly specific and could not be obtained with any other applied phospholipid.

Influence of surface charge on the incorporation and orientation of cytochrome c oxidase in liposomes.

Cytochrome c-oxidase is usually oriented 80-90% right-side-out when reconstituted with asolectin by the cholate dialysis method. Transformation of positively charged lysine groups at the matrix domain into negatively charged groups with succinic anhydride results in random orientation. A random orientation is also found after reconstitution in phosphatidylcholine, which can be changed into predominant right-side-out orientation by addition of cardiolipin. It is concluded that electrostatic interaction between positively charged groups of cytochrome c-oxidase with negative groups of phospholipids determines the asymmetric orientation of the enzyme in liposomes.

Effect of ionic strength on membrane potential generation in reconstituted cytochrome c oxidase.

In this study we applied the potential-sensitive fluorescent dye indocyanine to measure the initial rate of membrane potential development in liposomes containing reconstituted cytochrome c oxidase from bovine heart. A linear relationship was found between the initial rate of membrane potential development, related to the H+/e(-)-stoichiometry, and the rate of electron flow at various substrate concentrations. By increasing the salt concentration a decrease of the initial rate of membrane potential development was found. A similar dependence on the salt concentration was also found for the membrane potential under steady state conditions. The data further demonstrate variation of the H+/e(-)-stoichiometry in cytochrome c oxidase depending on the salt concentration in the buffer.

Influence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline modification on proton translocation and membrane potential of reconstituted cytochrome-c oxidase support "proton slippage".

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and modified with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ reacted mainly with subunits II and III and to a lower extent with subunit I, as shown by difference labeling with [14C]dicyclohexylcarbodiimide. EEDQ treatment of cytochrome-c oxidase vesicles influenced ferrocytochrome c-induced proton pumping by reducing maximally the H+/e- stoichiometry from 0.84 (control) to 0.24, but had only small effects on respiration, respiratory control ratio, and proton conductivity of the proteoliposomes. By titrating the reaction rate of the control and the modified cytochrome-c oxidase vesicles versus the membrane potential, as measured with a Ph3MeP+ electrode, saturation curves are obtained, which in both cases approach 225 mV. The ratios of electron transport rates of the two proton pumps at various membrane potentials decrease between 160 and 225 mV from about 2.2 to 1, indicating that the nonlinear flow/force relationship of these proton pumps is at least partly due to "slippage" of proton pumping.

Tissue-specific regulation of cytochrome c oxidase efficiency by nucleotides.

Cytochrome c oxidase from bovine heart and liver was reconstituted in liposomes in the absence or presence of nucleotides. Intraliposomal ADP, and to a smaller extent intraliposomal ATP, increased the respiratory activity of the heart but not of the liver isozyme under uncoupled but not under coupled conditions, leading to increased respiratory control ratios. In a preceding publication [Anthony, G., Reimann, A., & Kadenbach, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 1652-1656], the stimulatory effect of intraliposomal ADP could be related to interaction with the matrix domain of subunit VIa-h (heart type). The data suggest a regulatory effect of matrix nucleotides in heart and skeletal muscle mitochondria on the efficiency of energy transduction in COX.

Valinomycin binds stoichiometrically to cytochrome c oxidase and changes its structure and function.

Valinomycin binds to soluble and reconstituted cytochrome c oxidase (COX) in a stoichiometric manner, as shown by a spectral shift of the oxidized gamma-band. No spectral change is found with nigericin or 18-crown-6 and in the absence of potassium ions. Titration of the proton pumping activity of reconstituted COX with valinomycin reached a maximum of H+/e- - 0.73 after addition of 1 mole of valinomycin per mole of reconstituted COX. It is concluded that K+-translocation in proton-pumping COX vesicles occurs via enzyme-bound valinomycin.

The K(+)-ionophores nonactin and valinomycin interact differently with the protein of reconstituted cytochrome c oxidase.

The K(+)-ionophores valinomycin and nonactin induce a qualitatively identical change of the visible spectrum of isolated oxidized cytochrome c oxidase (red shift), but the amplitude is half with nonactin. Valinomycin, in the presence or absence of a protonophore, stimulates the respiration of the reconstituted enzyme to a higher extent than nonactin and results in a higher Km for cytochrome c. In contrast, nonactin causes a fivefold rate of proton conductivity across a liposomal membrane, after induction of a K(+)-diffusion potential. The data indicate that respiratory control by these antibiotics is not only due to degradation of a membrane potential, but rather to specific interaction with and modification of cytochrome c oxidase.

Effect of trypsin on the kinetic properties of reconstituted beef heart cytochrome c oxidase.

Isolated beef heart cytochrome c oxidase was reconstituted in liposomes by the cholate dialysis method with 85% of the binding site for cytochrome c oriented to the outside. Trypsin cleaved specifically subunit VIa and half of subunit IV from the reconstituted enzyme. The kinetic properties of the reconstituted enzyme were changed by trypsin treatment if measured by the spectrophotometric assay but not by the polarographic assay. It is concluded that subunit VIa and/or subunit IV participate in the electron transport activity of cytochrome c oxidase.

Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart.

Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.

Intraliposomal nucleotides change the kinetics of reconstituted cytochrome c oxidase from bovine heart but not from Paracoccus denitrificans.

Isolated cytochrome c oxidases of P. denitrificans and bovine heart were reconstituted in liposomes and the kinetics of cytochrome c oxidation were measured in the presence and absence of nucleotides either inside or outside of proteoliposomes, and after photolabelling with 8-azido-ATP. Intraliposomal ATP increases and ADP decreases the kinetics of ferrocytochrome c oxidation of the bovine but not of the Paracoccus enzyme. Extra-liposomal ATP and ADP increase the Km for cytochrome c of both enzymes, but ATP acts at lower concentrations than ADP. The increase of the Km for cytochrome c is obtained in coupled as well as in uncoupled proteoliposomes. Photolabelling with 8-azido-ATP of the reconstituted Paracoccus enzyme also increases the Km for cytochrome c which is completely prevented if ATP but not if ADP is present during illumination as was found with reconstituted cytochrome c oxidase from bovine heart. The data suggest a specific interaction of ATP and ADP with nuclear-coded subunits of bovine heart cytochrome c oxidase from the matrix side, because the effects are not found with the Paracoccus enzyme, which lacks these subunits.

ADP increases the affinity for cytochrome c by interaction with the matrix side of bovine heart cytochrome c oxidase.

The effect of intraliposomal ADP and ATP on the kinetics of cytochrome c oxidation in reconstituted bovine heart cytochrome c oxidase was measured by the photometric and polarographic method: 1. Intraliposomal ADP decreases and intraliposomal ATP increases the Km for cytochrome c when measured by the photometric assay under uncoupled conditions. 2. The above described effects are not obtained when the kinetics are measured with the polarographic assay. 3. Extraliposomal ATP increases the Km for cytochrome c similar to intraliposomal ATP, but this effect is measured with both methods of assay. 4. Under coupled conditions only a small decrease of the Km for cytochrome c by intraliposomal ADP is found.

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera.

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.

Proton slippage in cytochrome c oxidase of Paracoccus denitrificans. Membrane-potential measurements with the two-subunit and three-subunit enzyme.

Isolated cytochrome c oxidase from Paracoccus denitrificans, containing either two or three subunits, was reconstituted into liposomes and the membrane potential was measured at different rates of respiration using a triphenylmethylphosponium bromide electrode. Both enzymes revealed a non-linear increase of the membrane potential with increasing respiratory rates. The ratios of the respiratory rates of the two proton pumps decreased with increasing membrane potential, suggesting slippage of proton pumping, as has been shown before with two cytochrome c oxidases from bovine heart, differing in H+/e- stoichiometries due to chemical modification [Steverding, D. & Kadenbach, B. (1991) J. Biol. Chem. 266, 8097-8101]. The data suggest that slippage of proton pumping represents an intrinsic property of cytochrome c oxidase associated with the two catalytic subunits, I and II.

ATP and ADP bind to cytochrome c oxidase and regulate its activity.

By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes.

A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.