Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.

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Cytochrome c-mediated electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase. Kinetic evidence for a mobile cytochrome c pool

Biochemical Journal ◽

10.1042/bj2170551 ◽

1984 ◽

Vol 217 (2) ◽

pp. 551-560 ◽

Cited By ~ 16

Author(s):

R J Froud ◽

C I Ragan

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

State Level ◽

Complex Iii ◽

Molar Ratio ◽

Cytochrome C Reductase ◽

Molar Ratios ◽

Ubiquinol Oxidase ◽

Mediated Electron Transfer ◽

Pool Model

Ubiquinol oxidase has been reconstituted from ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase (Complex IV). The steady-state level of reduction of cytochrome c by ubiquinol-2 varies with the molar ratios of the complexes and with the presence of antimycin in a way that can be quantitatively accounted for by a model in which cytochrome c acts as a freely diffusible pool on the membrane. This model was based on that of Kröger & Klingenberg [(1973) Eur. J. Biochem. 34, 358-368] for ubiquinone-pool behaviour. Further confirmation of the pool model was provided by analysis of ubiquinol oxidase activity as a function of the molar ratio of the complexes and prediction of the degree of inhibition by antimycin.

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Cytochrome c mediates electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase by free diffusion along the surface of the membrane

Biochemical Journal ◽

10.1042/bj2170561 ◽

1984 ◽

Vol 217 (2) ◽

pp. 561-571 ◽

Cited By ~ 14

Author(s):

R J Froud ◽

C I Ragan

Keyword(s):

Phase Transition ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Rotational Diffusion ◽

Complex Iii ◽

Complex Iv ◽

Cytochrome C Reductase ◽

Ubiquinol Oxidase ◽

Spin Resonance ◽

Gel Phase

Ubiquinol oxidase can be reconstituted from ubiquinol-cytochrome c reductase (Complex III) and cytochrome c oxidase (Complex IV) whose endogenous phosphatidylcholine and phosphatidylethanolamine have been replaced by dimyristoylglycerophosphocholine. Phase transition of the lipid has no effect on Complex III and Complex IV activities assayed separately, but ubiquinol oxidase activity rapidly decreases as the temperature is lowered through the phase transition. A spin-labelled yeast cytochrome c derivative has been synthesized. Binding of the cytochrome c to liposomes demonstrates that only cardiolipin is involved under the conditions used for the ubiquinol oxidase experiments. In liposomes consisting of cardiolipin and dimyristoylglycerophosphocholine, e.s.r. (electron-spin-resonance) measurements show that rotational diffusion of cytochrome c is slowed in the gel phase of the latter lipid. We propose that the cytochrome c pool is bound to cardiolipin molecules, whose lateral and rotational diffusion in the bilayer is adequate to account for electron-transport rates.

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Observations on iron uptake, iron metabolism, cytochrome c content, cytochrome a content and cytochrome c-oxidase activity in regenerating rat liver

Biochemical Journal ◽

10.1042/bj0970532 ◽

1965 ◽

Vol 97 (2) ◽

pp. 532-539 ◽

Cited By ~ 16

Author(s):

ARL Gear

Keyword(s):

Cytochrome C ◽

Liver Regeneration ◽

Cytochrome C Oxidase ◽

Density Gradient ◽

Rat Liver ◽

Oxidase Activity ◽

Specific Content ◽

Cytochrome C Reductase ◽

Regenerating Rat Liver ◽

Mitochondrial Fractions

1. Differential and density-gradient centrifugation were used to fractionate mitochondria and fluffy layer from normal and regenerating rat liver. The iron, cytochrome a and cytochrome c contents and cytochrome c-oxidase activity were studied as well as the uptake of (59)Fe into protein and cytochrome c. 2. A certain degree of heterogeneity was evident between the heavy-mitochondrial and light-mitochondrial fractions, and in their behaviour during liver regeneration. 3. The specific content of light-mitochondrial iron and cytochrome a was 1.3-1.4 times that of heavy mitochondria. Changes in cytochrome c-oxidase activity closely followed those of cytochrome a content during liver regeneration, but not for light mitochondria after 10 days. 4. Radioactive iron ((59)Fe) was most actively taken up by well-washed light mitochondria during early liver regeneration. After 22 days fluffy layer became preferentially labelled. This substantiates the view that fluffy layer partially represents broken-down mitochondria. 5. During early regeneration, light-mitochondrial fractions separated along a density gradient were about 3 times as radioactive, and showed distinct heterogeneity of (59)Fe-labelling, in contrast with near homogeneity for heavy mitochondria. 6. Immediately after partial hepatectomy fractions corresponding to density 1.155 were 5-10 times as radioactive as particles of greater density. The radioactivity decreased sharply after 6 days. 7. These particles of low density possessed higher NADH-cytochrome c-reductase (1.5-5-fold) and succinate-dehydrogenase (1.1-2-fold) activities than typical mitochondrial fractions. Their succinate-cytochrome c-reductase and cytochrome c-oxidase activities were slightly lower. 8. The results are discussed in relation to mitochondrial morphogenesis, and a possible route from submitochondrial particles is suggested.

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Oxyradical production results from the Fe3+–doxorubicin complex undergoing self-reduction by its α-ketol group

Biochemistry and Cell Biology ◽

10.1139/o90-195 ◽

1990 ◽

Vol 68 (12) ◽

pp. 1331-1336 ◽

Cited By ~ 13

Author(s):

Brian B. Hasinoff

Keyword(s):

Cytochrome C ◽

Hydroxyl Radical ◽

Cytochrome C Oxidase ◽

Oxidase Activity ◽

Structural Difference ◽

Complex Iii ◽

Paramagnetic Resonance ◽

Radical Production ◽

Ferricytochrome C ◽

Resonance Spin

A variety of different measures have been used to compare the self-reduction of the Fe3+ complexes of doxorubicin and daunorubicin. The Fe3+ –doxorubicin complex exhibited a much faster rate of (i) O2 consumption, (ii) self-reduction under Ar to the Fe2+ complex, (iii) aerobic reduction of ferricytochrome c, (iv) scavenging of Fe2+ by bipyridine, (v) hydroxyl radical production measured by electron paramagnetic resonance spin-trapping experiments, and (vi) inactivation of the cytochrome c oxidase activity of beef heart submitochondrial particles, than did the corresponding Fe3+ –daunorubicin complex. In contrast to Fe3+ –doxorubicin, the Fe3+ –daunorubicin complex displayed only a fast phase of inhibition of the cytochrome c oxidase activity, indicating that the initial binding of these two Fe3+ –drug complexes is very similar. All of these results indicate that Fe3+ –doxorubicin undergoes a much faster self-reduction to the Fe2+ complex and hence a much greater rate of production of damaging oxyradicals when the Fe2+ is reoxidized by O2 or H2O2. The addition of the α-ketol acetol to Fe3+–daunorubicin resulted in greately increased rates of (i) ferricytochrome c reduction, (ii) Fe2+ production, and (iii) hydroxyl radical production. These results support the hypothesis that the α-ketol functional group of doxorubicin (which is not present on daunorubicin and is the only structural difference between these two compounds) reduces the Fe3+ while undergoing oxidation itself.Key words: doxorubicin, adriamycin, iron, self-reduction, oxyradical.

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An X linked (cardio) myopathy and granulocytopenia, with a low succinate cytochrome-c-reductase and cytochrome-c-oxidase activity in skeletal muscle

Clinical Neurology and Neurosurgery ◽

10.1016/0303-8467(84)90220-8 ◽

1984 ◽

Vol 86 (3) ◽

pp. 229

Author(s):

C.J. de Groot ◽

G.E. Beaufort-Krol ◽

W.F. Arts ◽

W. Blom ◽

M. Witsenburg ◽

...

Keyword(s):

Skeletal Muscle ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Oxidase Activity ◽

Cytochrome C Reductase ◽

Succinate Cytochrome C Reductase

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CYTOCHEMICAL STUDIES OF MITOCHONDRIA

The Journal of Cell Biology ◽

10.1083/jcb.2.6.653 ◽

1956 ◽

Vol 2 (6) ◽

pp. 653-669 ◽

Cited By ~ 84

Author(s):

Philip Siekevitz ◽

Michael L. Watson

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Adenylate Kinase ◽

Oxidase Activity ◽

Membrane Fraction ◽

Differential Centrifugation ◽

Active Fraction ◽

Mitochondrial Membranes ◽

Mitochondrial Preparation ◽

Cytochrome C Reductase

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ∼ 12 per cent of the protein N and ∼ 35 per cent of the phospholipide of the whole mitochondria and accounted for ∼ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.

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