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

1. The flux pathways for H+ and K+ movements into and out of proteoliposomes incorporating cytochrome c oxidase have been investigated as a function of the electrical and geometrical properties of the vesicles. 2. The respiration-induced pH gradient (ApH) and membrane potential (A/f) are mutually dependent and individually sensitive to the permeability properties of the membrane. A lowering or abolition of AVf by the addition of valinomycin increased the steady-state level of ApH. Conversely, removal of ApH by the addition of nigericin resulted in a higher steady-state AVf. 3. Vesicles prepared by sonication followed by centrifugation maintained similar pH gradients at steady state to those in vesicles prepared by dialysis, although the time taken to reach steady state was longer. Higher pH gradients can be induced in non-centrifuged sonicated preparations. 4. No significant differences were found in H+ and K+ permeability between proteoliposomes prepared by dialysis or by sonication. The permeability coefficient of the vesicle bilayers for H+ was 6.1 x 10-4 cm * s-' and that for K+ was 7.5 x 10-10 cm * s-1. An initial fast change in internal pH was seen on the addition of external acid or alkali, followed by a slower, ionophore-sensitive, change. The initial fast phase can be increased by the lipid-soluble base dibucaine and the weak acid oleate. In the absence of ionophores, increasing concentrations of oleate increased the rate of HI translocation to a level similar to that seen in the presence of nigericin. Internal alkalinization could also be induced by oleate upon the addition of potassium sulphate. 5. The initial, pre-steady-state and steady-state ApH and AV changes can be simulated using a model in which the enzyme responds to both ApH and ARf components of the protonmotive force. At steady state, the electrogenic entry of K+ is countered by electroneutral exit via a K+/H+ exchange. 6. The permeability coefficient, PH' calculated from HI flux under steady-state turnover conditions, was approx. 100 times higher than the corresponding 'passive' measurements of PH. Under conditions of oxidase turnover, the vesicles appear to be intrinsically more permeable to protons.

Section: Resultssupporting

confidence: 53%

The proteoliposomal steady state. Effect of size, capacitance and membrane permeability on cytochrome-oxidase-induced ion gradients

Wrigglesworth

Cooper

Sharpe

et al. 1990

“…Fairly recently, Shinitzky and co-workers (Corda et al, 1982) have also applied DPH to the study of the influence of a transmembrane potential on the lipid microviscosity of artificial phospholipid vesicles. Our investigation will attempt to extend these observations to natural membranes and possibly shed some light on the effects of electric fields on the integrity of the membranes of mitochondria and the phospholipid vesicles used for protein reconstitution studies (see, e.g., Casey et al, 1984). We applied the fluorescent probe DPH and spin-labelled fatty acids (see, e.g., Schreier et al, 1978) to measure lipid microviscosity.…”

mentioning

confidence: 99%

Membrane-potential-dependent changes of the lipid microviscosity of mitochondria and phospholipid vesicles

O'Shea¹,

Feuerstein-Thelen²,

Azzi³

1984

The effects of a transmembrane potential difference upon the lipid microviscosity of cytochrome oxidase vesicles (COVs) and rat liver mitochondria (RLM) were investigated. COVs and RLM were labelled with the fluorescent probe 1,6-diphenylhexa-1,3,5-triene (DPH). The fluorescence polarization of the probe was then measured when potentials of different magnitudes were induced across the membranes of these particles. It was shown that the absolute value of the microviscosity changes to quite a significant extent, owing to the imposition of large membrane potentials. On relaxation of the membrane potential the lipid microviscosity was also shown to return to the value before the induction of the potential. The largest change in lipid microviscosity was observed when coupled respiration was initiated. This occurred in both the COV system and the RLM system. The absolute value of the lipid microviscosity was shown to change by as much as 22% with the induction of membrane potentials, owing to respiration. To confirm the viscosity measurements made with DPH, lipid microviscosity was also measured with the spin-labelled fatty acid 5-doxyl stearate. Measurements of the order parameters indicated that, in agreement with the results of fluorescence experiments, viscosity changes occurred that were due to the induction of a membrane potential. The significance of these findings to the regulation of metabolism is briefly discussed, the main conclusion being that, although there is certainly a significant variation of lipid microviscosity with electric field, mechanistic interpretations will require further studies.

“…(1) Owing to the use of an insufficient chargecompensation system (Casey et al, 1979), a significant transmembrane potential may have remained (Casey et al, 1984) with increasing amounts of ferrocytochrome c added, through which respiratory control on the COVs occurred.…”

Section: Resultsmentioning

confidence: 99%

“…The aerobic oxidation of ferrocytochrome c by reconstituted ox heart cytochrome c oxidase (EC 1.9.3.1) is accompanied by a transient acidification of the external medium followed by a persistent net alkalinization of the system (Hinkle et al, 1972;Wikstrom & Saari, 1977;Casey et al, 1979Casey et al, , 1984Coin & Hinkle, 1979;Prochaska et al, 1981;Papa et al, 1983;Proteau et al, 1983;Thelen et al, 1985). Although it is generally agreed that the reduction of oxygen by cytochrome c oxidase involves the net consumption of one proton from the intravesicular space per externally oxidized ferrocytochrome c molecule, the nature of the observed transient acidification of the external medium remains a matter of some dispute (see, for review, Mitchell & Moyle, 1983;Casey & Azzi, 1983).…”

mentioning

confidence: 99%

“…an acidor base-induced ApH or a respiration-induced pH gradient (Krab & Wikstrom, 1978;Casey et al, 1979). This difference has been proved to be in part due to an inadequate charge-compensation system (Casey et al, 1984). The second is related to the change of the H+/e--ratio when higher numbers of turnovers are initiated (Casey et al, 1979;Proteau et al, 1983).…”

mentioning

confidence: 99%

See 1 more Smart Citation

New insights on the cytochrome c oxidase proton pump

Thelen¹,

O'Shea²,

Azzi³

1985

Cytochrome c oxidase vesicles were used to show that, under appropriate experimental conditions: (1) no net deprotonation of the vesicular membrane or of the incorporated enzyme occurs during the oxidation of ferrocytochrome c; (2) the pH equilibration kinetics of a respiration-induced pH gradient across the bilayer are a simple function of the ohmic proton-conductance properties of the membrane; (3) a fairly constant stoichiometry (0.8-0.7) of the numbers of protons pumped per molecule of ferrocytochrome c oxidized, i.e. the H+/e- ratio, over a wide range of dioxygen molecules reduced (1-12) is observed.