The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association

1. The NADH-ubiquinone oxidoreductase complex (Complex I) and the ubiquinol-cytochrome c oxidoreductase complex (Complex III) combine in a 1:1 molar ratio to give NADH-cytochrome c oxidoreductase (Complex I-Complex III). 2. Experiments on the inhibition of the NADH-cytochrome c oxidoreductase activity of mixtures of Complexes I and III by rotenone and antimycin indicate that electron transfer between a unit of Complex I-Complex III and extra molecules of Complexes I or III does not contribute to the overall rate of cytochrome c reduction. 3. The reduction by NADH of the cytochrome b of mixtures of Complexes I and III is biphasic. The extents of the fast and slow phases of reduction are determined by the proportion of the total Complex III specifically associated with Complex I. 4. Activation-energy measurements suggest that the structural features of the Complex I-Complex III unit promote oxidoreduction of endogenous ubiquinone-10.

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The effects of lipid phase transitions on the interaction of mitochondrial NADH–ubiquinone oxidoreductase with ubiquinol–cytochrome c oxidoreductase

Biochemical Journal ◽

10.1042/bj1780415 ◽

1979 ◽

Vol 178 (2) ◽

pp. 415-426 ◽

Cited By ~ 26

Author(s):

C Heron ◽

M G Gore ◽

C I Ragan

Keyword(s):

Cytochrome C ◽

Cytochrome B ◽

Complex I ◽

Complex Iii ◽

Molar Ratio ◽

Lipid Phase ◽

Oxidoreductase Activity ◽

Arrhenius Plots ◽

Nadh Cytochrome ◽

Ubiquinone 10

1. The endogenous phosphatidylcholine and phosphatidylethanolamine of Complexes I and III from bovine heart mitochondria may be completely replaced with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine with at least partial retention of activity. 2. The lipid-replaced enzymes associate in 1:1 molar ratio to give a Complex I–III unit catalysing NADH-cytochrome c oxidoreductase activity. 3. On increasing the concentration of ubiquinone-10 and the synthetic phospholipid, the lipid-replaced Complexes appear to operate independently of each other as in the natural membrane. Thus the lipid-replaced enzymes associate in exactly the same ways as the enzymes containing natural phospholipids. 4. Arrhenius plots of NADH–cytochrome c oxidoreductase activity reconstituted from lipid-replaced Complexes I and III exhibit changes in slope at 24 degrees C. When the concentrations of phospholipid and ubiquinone-10 are increased, the Arrhenius plots show discontinuities at 24 degrees C as well as changes in slope. 5. The kinetics of cytochrome b reduction by NADH were measured in mixtures containing 2 mol of Complex III/mol of Complex I. When the enzymes contained natural phospholipids. the reduction kinetics were biphasic. When the enzymes had been supplemented with further phospholipid and ubiquinone-10 the kinetics were monophasic. When lipid-replaced enzymes were supplemented with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine and ubiquinone-10, reduction of cytochrome b was monophasic above the phase-transition temperature of the lipid but biphasic below it. 6. These findings are interpreted in terms of the model for the interaction of Complexes in the natural membrane proposed by Heron, Ragan & Trum-power [(1978) Biochem. J. 174, 791–800].

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Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide–adenine dinucleotide–ubiquinone oxidoreductase from bovine heart mitochondria

Biochemical Journal ◽

10.1042/bj1650295 ◽

1977 ◽

Vol 165 (2) ◽

pp. 295-301 ◽

Cited By ~ 11

Author(s):

Susan E. Crowder ◽

C. Ian Ragan

Keyword(s):

Complex I ◽

Time Course ◽

Nadh Dehydrogenase ◽

Sodium Dodecyl ◽

Complex Iii ◽

Type Ii ◽

Oxidoreductase Activity ◽

Ubiquinone Oxidoreductase ◽

Nadh Cytochrome ◽

Reduced Nicotinamide Adenine Dinucleotide

1. Incubation of NADH–ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH–K3Fe(CN)6 oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH–cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH–K3Fe(CN)6 oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.

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The effects of lipid fluidity on the rotational diffusion of complex I and complex III in reconstituted NADH-cytochrome c oxidoreductase

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(82)90477-1 ◽

1982 ◽

Vol 693 (1) ◽

pp. 113-124 ◽

Cited By ~ 10

Author(s):

Veronica M. Poore ◽

John T.R. Fitzsimons ◽

C. Ian Ragan

Keyword(s):

Cytochrome C ◽

Complex I ◽

Rotational Diffusion ◽

Complex Iii ◽

Lipid Fluidity ◽

Nadh Cytochrome

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The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Restoration of ubiquinone-pool behaviour

Biochemical Journal ◽

10.1042/bj1740791 ◽

1978 ◽

Vol 174 (3) ◽

pp. 791-800 ◽

Cited By ~ 67

Author(s):

C Heron ◽

C I Ragan ◽

B L Trumpower

Keyword(s):

Electron Transfer ◽

Electron Transport ◽

Complex I ◽

Mitochondrial Membrane ◽

Complex Iii ◽

Relative Mobility ◽

Inner Mitochondrial Membrane ◽

Ubiquinone Oxidoreductase ◽

Nadh Ubiquinone Oxidoreductase ◽

Ubiquinone 10

1. In the inner mitochondrial membrane, dehydrogenases and cytochromes appear to act independently of each other, and electron transport has been proposed to occur through a mobile pool of ubiquinone-10 molecules [Kröger & Klingenberg (1973) Eur. J. Biochem. 34, 358–368]. 2. Such behaviour can be restored to the interaction between purified Complex I and Complex III by addition of phospholipid and ubiquinone-10 to a concentrated mixture of the Complexes before dilution. 3. A model is proposed for the interaction of Complex I with Complex III in the natural membrane that emphasizes relative mobility of the Complexes rather than ubiquinone-10. Electron transfer occurs only through stoicheiometric Complex I-Complex III units, which, however, are formed and re-formed at rates higher than the rate of electron transfer.

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