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

ABSTRACT: 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].

SUBMITTER: Heron C

PROVIDER: S-EPMC1186530 | biostudies-other | 1979 Feb

REPOSITORIES: biostudies-other

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Project description:The mode of action of Deltalac-acetogenins, strong inhibitors of bovine heart mitochondrial complex I, is different from that of traditional inhibitors such as rotenone and piericidin A [Murai, M., et al. (2007) Biochemistry 46 , 6409-6416]. As further exploration of these unique inhibitors might provide new insights into the terminal electron transfer step of complex I, we drastically modified the structure of Deltalac-acetogenins and characterized their inhibitory action. In particular, on the basis of structural similarity between the bis-THF and the piperazine rings, we here synthesized a series of piperazine derivatives. Some of the derivatives exhibited very potent inhibition at nanomolar levels. The hydrophobicity of the side chains and their balance were important structural factors for the inhibition, as is the case for the original Deltalac-acetogenins. However, unlike in the case of the original Deltalac-acetogenins, (i) the presence of two hydroxy groups is not crucial for the activity, (ii) the level of superoxide production induced by the piperazines is relatively high, (iii) the inhibitory potency for the reverse electron transfer is remarkably weaker than that for the forward event, and (iv) the piperazines efficiently suppressed the specific binding of a photoaffinity probe of natural-type acetogenins ([ (125)I]TDA) to the ND1 subunit. We therefore conclude that the action mechanism of the piperazine series differs from that of the original Deltalac-acetogenins. The photoaffinity labeling study using a newly synthesized photoreactive piperazine ([ (125)I]AFP) revealed that this compound binds to the 49 kDa subunit and an unidentified subunit, not ND1, with a frequency of approximately 1:3. A variety of traditional complex I inhibitors as well as Deltalac-acetogenins suppressed the specific binding of [ (125)I]AFP to the subunits. The apparent competitive behavior of inhibitors that seem to bind to different sites may be due to structural changes at the binding site, rather than occupying the same site. The meaning of the occurrence of diverse inhibitors exhibiting different mechanisms of action is discussed in light of the functionality of the membrane arm of complex I.

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

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