Lipid requirements for cytochrome c oxidase activity.

Cytochrome c oxidase depleted of endogenous lipid by detergent exchange has been reconstituted into vesicles with synthetic lipids of known head group and fatty acid composition and enzymic activities have been measured. No evidence for head group specificity was found. However, the enzyme does require the fluid environment provided by unsaturated fatty acids. The state of dispersion of the enzyme was found to affect the activities regenerated in reconstitution studies. The highest activities were obtained using lysolecithin containing an oleoyl fatty acid as the lipid component.     

Cation transport in cytochrome oxidase reconstituted vesicles.

Cation translocation across the membrane of cytochrome oxidase reconstituted vesicles may be followed with a simple spectrophotometric method. Cytochrome oxidase reconstituted vesicles, supplemented with ascorbate and cytochrome c. induce large spectral changes of the positive dye safranine, reversed by uncouplers and inhibitors of respiration. The dye is probably accumulated in the inner space of the vesicles, where it reaches high concentrations and aggregates. The spectral shifts and the absorbance changes, due to aggregation, are proportional to the amount of the dye taken up and depend on the respiratory control. In the presence of potassium, valinomycin causes an inhibition, whereas nigericin stimulates the dye uptake. The data are discussed in terms of electrical potential dependent fluxes.     

Intrinsic uncoupling in proton-pumping cytochrome c oxidase: pH dependence of cytochrome c oxidation in coupled and uncoupled phospholipid vesicles

The pH dependence of the transient aerobic kinetics of cytochromes c and a has been investigated with cytochrome oxidase reconstituted in phospholipid vesicles in the absence and presence of an uncoupler and an ionophore. The cytochrome a reduction level immediately after the burst phase was 60-80% and was not significantly changed by the addition of uncoupler and/or ionophore. The coupled rate of ferro-cytochrome c oxidation increases linearly with decreasing pH in the range 8.4-5.4. The increase in rate on uncoupling becomes less with decreasing pH and low cytochrome c concentration, being almost zero at pH 5.4. The coupled rate is increased by a lowering of the outside pH when the inside pH is constant. Varying the inside pH with a constant outside pH of 7.4 has little effect on the rate. It is suggested that the electrochemical potential has two separate effects on the coupled rate: the pH gradient mainly slows down the intramolecular electron transfer, but the membrane potential also lowers the second-order rate constant for the reaction with cytochrome c. The results are interpreted in terms of a model in which protonation of an acid-base group with a pKa of 6.4 from the inside increases the catalytic constant. Protonation from the outside, on the other hand, leads to an intrinsic uncoupling, because the protonated enzyme in the output state can return to the input state. This has no adverse physiological effect, since it becomes significant only at pH values well below 7.     

Rotational motion of cytochrome c oxidase in phospholipid vesicles

Natural abundance ¹³C and high field ¹H NMR spectroscopy are used to characterize the major coat protein of the filamentous bacteriophage fd in sodium dodecyl sulfate micelles. Chemical shift dispersion of protein resonances, slow and differential exchange rates of amide protons, and relaxation parameters of the alpha carbons of the protein indicate that the detergent solubilized coat protein has a stable native conformation. The structure of the coat protein in micelles differs from that found for typical globular proteins in solution in that parts of the peptide backbone exhibit rapid segmental motion.     

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H⁺ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K⁺. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H⁺ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H⁺.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Quantization of membrane potential generation by cytochrome c oxidase in small vesicles

Proteoliposomes incorporating cytochrome c oxidase have been prepared by the cholate dialysis method and by sonication. Sonication produces multilamellar vesicles heterogeneous in size in contrast to a more uniform preparation of unilamellar vesicles produced by the dialysis procedure. Respiratory control in both preparations ranges between 4 and 8. From an electron microscopic analysis of proteoliposome size, the average electrical capacitance/vesicle for the dialyzed and sonicated preparations is calculated as 15 X 10(-18) F and 130 X 10(-18) F, respectively. These capacitance values would lead to a quantization of membrane potential generation by the enzyme at 77 mV/turnover for the dialyzed preparation and 9 mV/turnover for sonicated vesicles. It is argued that these differences can explain the dependence of H+ translocation on the number of turnovers of cytochrome c oxidase in dialyzed preparations in contrast to the lack of dependence on number of turnovers in sonicated preparations.     

Studies on the transmembrane orientation of cytochrome c oxidase in phospholipid vesicles

We report investigations into the direction of orientation of cytochrome c oxidase in reconstituted vesicles and the factors determining this. Measurement of the enzyme orientation employed two independent techniques: monitoring of the level of haem reduction by membrane-permeant and membrane-impermeant reagents and a kinetic analysis of the reduction of a spin label covalently bound to the oxidase surface. The method of preparation of the oxidase vesicles had a pronounced effect on the enzyme orientation and the two measurement techniques agreed in indicating that the proportion of mitochondrially oriented enzyme was approximately 85% and 50% for vesicles prepared by cholate dialysis and sonication respectively. Our results show that the membrane orientation of the oxidase is determined by interactions between the phospholipid bilayer and the portion of the enzyme embedded therein, as opposed to gross physical constraints. In particular, we demonstrate that the orientation of the oxidase is affected by the fluidity and surface charge of the membrane.     

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.     

Reconstitution of cytochrome c oxidase in phospholipid vesicles containing polyvinylic polymers.

Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368].     

A quantitative characterisation of H+ translocation by cytochrome c oxidase vesicles

A quantitative analysis of H+ extrusion by reconstituted cytochrome c oxidase vesicles is presented with particular regard to the decay kinetics of the extruded proton pulse and to the structural heterogeneity of the vesicle preparation. The decay of the extruded H+ pulse under conditions typical of those used for its measurement is much slower than expected from the passive proton permeability of the vesicle membranes. It is shown that this apparent anomaly results from insufficient transmembrane charge equilibration via valinomycin and K+ during oxidase turnover. This situation can be remedied by increasing the valinomycin concentration or by replacing this counterion system with 1 mM tetraphenylphosphonium. Under these latter conditions, the decay kinetics can be described as the sum of two exponential terms. To facilitate interpretation of the proton pump decay kinetics, a structural analysis of the oxidase vesicle preparation is presented. The bulk of the reconstituted vesicles (i.e., those representing approx. 80% of the total oxidase and lipid) are 30-62 nm in diameter. At least 70% of the reconstituted oxidase molecules are contained individually in separate vesicles, indicating that the enzyme monomer is competent in H+ translocation.     

Variations on the "dilution" method for reconstituting cytochrome c oxidase into membrane vesicles

A method for the rapid incorporation of cytochrome c oxidase into membranes has been developed. This method essentially consists of obtaining a preparation of the enzyme in which it is isolated and then dissolving it in a medium containing 0.5% of the detergent Tween 20, which gives a final concentration of 0.0125% after reconstitution. These studies revealed an optimal ratio of 1 microgram of enzyme to 5 mg of phospholipids. A similar optimal ratio was found when the amount of protein was varied. The optimum temperature was found to be 30 degrees C. Without a peak value being reached, it was found that the best reconstitution was obtained at pH 7.0-8.0. When measurements were performed either with a fluorescent cyanine (DiSC3) or by the uptake of tetraphenylphosphonium, it was found that the enzyme, with cytochrome c added to the outside, was capable of generating a membrane potential that was negative inside. Using the same procedure, the enzyme could also be reconstituted into vesicles of yeast plasma membrane. The procedure, then, seems adequate for incorporating cytochrome c oxidase into different kinds of membrane vesicles.     

Preparation of reconstituted cytochrome oxidase vesicles with defined trans-membrane protein orientations employing a cytochrome c affinity column

Reconstituted cytochrome oxidase systems in which the majority of the vesicles contain a single oxidase dimer can be prepared. It is shown that, when these are passed through a cytochrome c affinity column, only those vesicles oriented outwards (such that the active site is available to external cytochrome c) are bound to the support matrix. Protein-free vesicles and vesicles containing an inwardly oriented enzyme are eluted in the void volume. Subsequently, vesicles containing an outwardly oriented enzyme can be eluted from the column at high salt concentrations. This protocol has been used successfully to resolve vesicles of either oxidase orientation when the enzyme is reconstituted with a variety of lipid mixtures. The recovery of oxidase activity from the column ranged between 75 and 94%.     

Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.     

Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle.2. The activity of cytochrome c oxidase in the reconstituted vesicles is not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c.3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH^? inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externally added ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.