Dicyclohexylcarbodiimide inhibition of succinate- and ubiquinol-cytochrome c reductase in beef heart mitochondria

We have found that dicyclohexylcarbodiimide (DCCD) inhibits both the succinate-cytochrome c and the ubiquinol-cytochrome c reductases in cytochrome c-depleted mitochondria. On the other hand the succinate-ubiquinone reductase is not decreased at the same levels of the inhibitor. The inhibition curve of DCCD results sigmoidal for succinate-cytochrome c reductase, whereas it is hyperbolic for the ubiquinol-1-cytochrome c reductase, with also a lower apparent KI. The inhibition appears dependent both on the time of preincubation and on the mitochondrial concentration. The apparent Km for ubiquinol-1 is increased and the maximal velocity of ubiquinol-cytochrome c reductase is decreased by DCCD. The effects do not appear to be caused by unspecific modification of the physicochemical state of the bc1 region of the respiratory chain. The results therefore suggest the presence of a DCCD-sensitive electron transfer step in the redox pathways from ubiquinol to cytochrome c.     

Structural and functional alterations induced in the mitochondrial cytochrome b-c1 complex by N,N'-dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstituted mitochondrial cytochrome b-c1 complex. In proteoliposomes containing b-c1 complex DCCD inhibits equally electron flow and proton translocation catalyzed by the enzyme. In both isolated and reconstituted systems the inhibitory effect is accompanied by structural alterations in the polypeptide pattern of the enzyme consistent with cross-linking between subunits V and VII. The kinetics of inhibition of enzymic activity correlates with that of the cross-linking, suggesting that the two phenomena may be coupled. Binding of [14C] DCCD to both isolated and reconstituted enzyme was also observed, though it was not correlated kinetically with the inhibition.     

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c1, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c1 complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c1 complex. The binding of [14C]DCCD to the isolated b-c1 complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c1 complex are discussed.     

Phospholipid-dependent interaction between dibromothymoquinone and iron-sulfur protein in mitochondrial ubiquinol-cytochrome c reductase

Dibromothymoquinone (DBMIB) inhibits antimycin A-sensitive ubiquinol-cytochrome c reductase activity; the maximal inhibition is 90%. DBMIB alters the EPR spectra of reduced iron-sulfur protein in intact ubiquinol-cytochrome c reductase. The maximal spectral change occurs with 60 mol inhibitor per mol cytochrome c1 in the reductase. DBMIB causes little alteration in the EPR characteristics of iron-sulfur protein when ubiquinol-cytochrome c reductase is delipidated. When delipidated ubiquinol-cytochrome c reductase is replenished with phospholipid, the effect of DBMIB reappears. However, when DBMIB is added to delipidated protein prior to replenishment with phospholipid, very little spectral alteration is observed. DBMIB does not alter the EPR spectra of purified iron-sulfur protein, with or without phospholipid in the preparation. Reduced DBMIB does not alter the EPR characteristics of iron-sulfur protein in intact or delipidated ubiquinol-cytochrome c reductase. Cysteine and other thiol compounds can reverse the spectral alternation caused by DBMIB. This reversal probably results from the reduction of DBMIB.     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide

N,N′-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c₁, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c₁ complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c₁ complex. The binding of [¹⁴C]DCCD to the isolated b-c₁ complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c₁ complex are discussed.     

Triple inhibitor titrations support the functionality of the dimeric character of mitochondrial ubiquinol-cytochrome c oxidoreductase.

The ubiquinol-2 or duroquinol oxidoreductase activity of mitochondrial ubiquinol-cytochrome c oxidoreductase was titrated with combinations of antimycin, myxothiazol and N,N'-dicyclohexylcarbodiimide (DCCD). A statistical model has been developed that can predict the activity of the complex treated with all possible combinations of these inhibitors. On the basis of the measured titration curves the model had to accommodate interaction between the two promoters of the complex. The titrations confirm that treatment with DCCD results in the modification of a certain site in one of the two promoters of the bc1 dimer, thereby blocking one antimycin A binding site without inhibiting electron transfer. Modification of both antimycin A binding sites of the dimer is apparently required for inhibition of electron transfer through the complex, just as modification of both myxothiazol-binding sites is required for full inhibition. The conclusion can be drawn that mitochondrial ubiquinol-cytochrome c oxidoreductase is a functional dimer, consisting of electrically interacting protomers.     

Phospholipid-dependent interaction between dibromothymoquinone and iron-sulfur protein in mitochondrial ubiquinol-cytochrome c reductase

(1) Dibromothymoquinone (DBMIB) inhibits antimycin A-sensitive ubiquinol-cytochrome c reductase activity; the maximal inhibition is 90%. (2) DBMIB alters the EPR spectra of reduced iron-sulfur protein in intact ubiquinol-cytochrome c reductase. The maximal spectral change occurs with 60 mol inhibitor per mol cytochrome c₁ in the reductase. (3) DBMIB causes little alteration in the EPR characteristics of iron-sulfur protein when ubiquinol-cytochrome c reductase is delipidated. (4) When delipidated ubiquinol-cytochrome c reductase is replenished with phospholipid, the effect of DBMIB reappears. However, when DBMIB is added to delipidated protein prior to replenishment with phospholipid, very little spectral alteration is observed. (5) DBMIB does not alter the EPR spectra of purified iron-sulfur protein, with or without phospholipid in the preparation. (6) Reduced DBMIB does not alter the EPR characteristics of iron-sulfur protein in intact or delipidated ubiquinol-cytochrome c reductase. (7) Cysteine and other thiol compounds can reverse the spectral alternation caused by DBMIB. This reversal probably results from the reduction of DBMIB.     

ATP-dependent proton translocation and quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of a cytochrome-deficient mutant of Escherichia coli,.

1. ATP-dependent proton translocation and ATP-dependent quenching of the fluorescence of 9-aminoacridine were measured in inside-out vesicles derived from a cytochrome-deficient mutant of Escherichia coli. 2. ATP-dependent quenching of fluorescence was inhibited by nigericin gramicidin, NH4Cl, and carbonylcyanide-m-chlorophenylhydrazone. Inhibition was also produced by the ATPase inhibitors N,N'-dicyclohexylcarbodimide (DCCD) and diphenyl phosphorazidate (DPA), and by the respiratory chain inhibitors piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and An2+. The inhibition of ATP-dependent fluorescence quenching by the ionophores, uncouplers, and respiratory chain inhibitors was not due to an effect on ATPase activity which was insensitive to these agents. 3. By use of the ATPase inhibitors DCCD and DPA, or by replacing ATP with GTP, ITP and CTP, a correlation between the ATPase activity and the rate of ATP-dependent membrane energization, as measured by fluorescence quenching, was obtained.     

Effect of substituents of the benzoquinone ring on electron-transfer activities of ubiquinone derivatives

The effect of substituents on the 1,4-benzoquinone ring of ubiquinone on its electron-transfer activity in the bovine heart mitochondrial succinate-cytochrome c reductase region is studied by using synthetic ubiquinone derivatives that have a decyl (or geranyl) side-chain at the 6-position and various arrangements of methyl, methoxy and hydrogen in the 2, 3 and 5 positions of the benzoquinone ring. The reduction of quinone derivatives by succinate is measured with succinate-ubiquinone reductase and with succinate-cytochrome c reductase. Oxidation of quinol derivatives is measured with ubiquinol-cytochrome c reductase. The electron-transfer efficacy of quinone derivatives is compared to that of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone. When quinone derivatives are used as the electron acceptor for succinate-ubiquinone reductase, the methyl group at the 5-position is less important than are the methoxy groups at the 2- and 3-positions. Replacing the 5-methyl group with hydrogen causes a slight increase in activity. However, replacing one or both of 2- and 3-methoxy groups with a methyl completely abolishes electron-acceptor activity. Replacing the 3-methoxy group with hydrogen results in a complete loss of electron-acceptor activity, while replacing the 2-methoxy with hydrogen results in an activity decrease by 70%, suggesting that the methoxy group at the 3-position is more specific than that at the 2-position. The structural requirements for quinol derivatives to be oxidized by ubiquinol-cytochrome c reductase are less strict. All 1,4-benzoquinol derivatives examined show partial activity when used as electron donors for ubiquinol-cytochrome c reductase. Derivatives that possess one unsubstituted position at 2, 3 or 5, with a decyl group at the 6-position, show substrate inhibition at high concentrations. Such substrate inhibition is not observed when fully substituted derivatives are used. The structural requirements for quinone derivatives to be reduced by succinate-cytochrome c reductase are less specific than those for succinate-ubiquinone reductase. Replacing one or both of the 2- and 3-methoxy groups with a methyl and keeping the 5-position unsubstituted (plastoquinone derivatives) yields derivatives with no acceptor activity for succinate-Q reductase. However, these derivatives are reducible by succinate in the presence of succinate-cytochrome c reductase. This reduction is antimycin-sensitive and requires endogenous ubiquinone, suggesting that these (plastoquinone) derivatives can only accept electrons from the ubisemiquinone radical at the Qi site of ubiquinol-cytochrome c reductase, and cannot accept electrons from the QPs of succinate-ubiquinone reductase.     

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrialbc 1 complex

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrialbc ₁ complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in thebc ₁ complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochromec reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of thebc ₁ complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochromec reductase activity. However, a clear stimulation of the rate of cytochromeb reduction in parallel to an inhibition of cytochromeb oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on thebc ₁ complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.     

Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [14C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [14C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity.     

The nature of the inhibition of 4,7-dioxobenzothiazole derivatives on mitochondrial ubiquinol-cytochrome c reductase

To investigate the inhibitory action and binding site of a quinone-like molecule, 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), a series of 4,7-dioxobenzothiazole derivatives were synthesized and their inhibitory efficiencies studied. Replacing the 6-hydroxyl or 2-hydrogen of UHDBT with a bromo or a methoxy group causes only a slight decrease in inhibitory efficiency, indicating that the 6-hydroxyl or the 2-hydrogen of UHDBT is not a structural requirement for inhibition. 5-Undecyl-6-bromo (or methoxy)-4,7-dioxobenzothiazole shows a pH-dependent inhibition similar to that observed with UHDBT, suggesting that the pH dependence is due to the presence of a dissociable group in the protein complex and not to the deprotonation of the hydroxyl group of the inhibitor. Replacing the 6-hydroxyl group with an azido group causes changes similar to those observed with UHDBT; the inhibition is accompanied by alteration of the epr characteristics of reduced iron-sulfur protein in ubiquinol-cytochrome c reductase. The extent of inhibition is not changed upon illumination of the treated reductase. When the photolyzed, 6-azido-5-(1',2'-[3H] undecyl)-4,7-dioxobenzothiazole [( 3H]6-azido-UDBT)-treated reductase is subjected to organic solvent extraction, no radioactivity is found in the reductase protein. Rather, the radioactivity is located in the phospholipid fraction. A [3H]azido-UDBT-cardiolipin adduct, identified after separation of the phospholipid fraction by high performance liquid chromatography, has 6-azido-UDBT linked to an acyl group, not to the head group of the cardiolipin molecule. These results suggest that inhibition by UHDBT is due to perturbation of specific cardiolipin molecules in ubiquinol-cytochrome c reductase. Since UHDBT and 6-azido-UDBT also inhibit the ubiquinol-cytochrome c reductase activity of delipidated reductase (10% of the original lipid remaining) assayed after reconstitution with ubiquinone and phospholipid, and the [3H]azido-UDBT-cardiolipin adduct is also found in the delipidated reductase, the UHDBT-perturbed cardiolipin molecule is structurally indispensable to reductase and it tightly bound to the reductase protein, most likely the quinone binding proteins.     

Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei

Interaction of N,N′-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30–65% inactivation over a concentration range of 5–50 μM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5·10⁵ M^?1·min^?1. The correlation between the initial binding of [¹⁴C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F₀F₁-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [¹⁴C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K⁺-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F₀F₁-ATPase complex.     

Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.

A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Sch?gger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.     

Interaction and identification of ubiquinone-binding proteins in ubiquinol-cytochrome c reductase by azido-ubiquinone derivatives

Various azido-ubiquinone derivatives were synthesized and characterized. 3-Azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone was found to be suitable for the study of specific interaction between ubiquinone (Q) and protein. It was synthesized with high specific radioactivity and used to identify the Q-binding proteins in purified ubiquinol-cytochrome c reductase. This azido-Q derivative showed partial efficiency in restoring activity to the Q- and phospholipids-depleted ubiquinol-cytochrome c reductase in the absence of light. Azido-Q derivative treated samples, however, became completely inactivated upon photolysis, and the inactivation was not reversed by addition of Q derivatives. The redox state of the azido-Q derivative has little effect on the Q-binding affinity. Two protein subunits with Mr = 37,000 and 17,000 were found to be heavily labeled when depleted ubiquinol-cytochrome c reductase was treated with [3H] azido-Q derivative followed by photolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of radioactive labeling of the Mr = 17,000 protein was proportional to the degree of inactivation and affected by the presence of phospholipids. The radioactive labeling of the Mr = 37,000 protein subunit, however, showed no correlation with degree of inactivation and was not affected by phospholipids. Since the radiolabeling at the Mr = 17,000 protein subunit was affected by phospholipids and correlated with the enzymatic activity, this subunit is probably the Q-binding protein in this enzyme complex (QPc). The inhibition of enzymatic activity by n-heptyl-4-hydroxyquinoline-N-oxide was easily reversed by addition of the azido-Q derivative. The distribution of radioactivity among the subunits of ubiquinol-cytochrome c reductase was not affected by the presence of antimycin A, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole or n-heptyl-4-hydroxyquinoline-N-oxide, suggesting that the binding site(s) of these inhibitors are not the Q-binding site.     

Dicyclohexylcarbodiimide blocks proton ejection and affects antimycin binding but not electron transport in complex III from yeast mitochondria

Treatment of complex III with dicyclohexyldicarbodiimide (DCCD) either before or after incorporation into liposomes resulted in a loss of electrogenic proton movements; however, only minimal decreases in cytochrome c reductase activity were noted in the liposomes containing DCCD-treated complex III. Thus, DCCD appears to act by "uncoupling" proton translocation from electron transport. A decreased sensitivity of the ubiquinol:cytochrome c reductase activity to antimycin was also noted in the DCCD-treated complex III. This loss of sensitivity to antimycin was reflected in a decreased binding of antimycin to the complex after DCCD treatment from 9.5 nmol/mg of protein in the control to 3.8 nmol/mg of protein in the DCCD-treated complex. DCCD also affected the red shift observed after antimycin addition to dithionite-reduced complex III resulting in a broad peak with no sharp maximum. Similarly, DCCD treatment of yeast mitochondria resulted in a complete loss in the red shift after antimycin addition to mitochondria previously reduced with succinate. No loss in enzymatic activity was observed in the DCCD-treated mitochondria. These results suggest that DCCD concomitant with the inhibition of proton ejection in the cytochrome b-c1 region of the respiratory chain causes modifications in the properties of cytochrome b which alter the binding of antimycin without significantly affecting the electron transfer activity of this cytochrome.     

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial succinate-ubiquinone and ubiquinol-cytochrome c reductases

The interaction between succinate-ubiquinone and ubiquinol-cytochrome c reductases in the purified, dispersed state and in embedded phospholipid vesicles was studied by differential scanning calorimetry and by electron paramagnetic resonance (EPR). When the purified, detergent-dispersed succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase undergo thermodenaturation, they show an endothermic transition. However, when these isolated electron-transfer complexes are embedded in phospholipid vesicles, they undergo exothermodenaturation. The energy released could result from the collapse of the strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both succinate-ubiquinone and ubiquinol-cytochrome c reductases was smaller than that of a mixture of protein-phospholipid vesicles formed from the individual electron-transfer complexes. This suggests specific interaction between succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase in the membrane. This idea is supported by saturation transfer EPR studies showing that the rotational correlation time of spin-labeled ubiquinol-cytochrome c reductase is increased when mixed with succinate-ubiquinone reductase prior to embedding in phospholipid vesicles. These results indicate that succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase are indeed present in the membrane as a supermacromolecular complex. No such supermacromolecular complex is detected between NADH-ubiquinone and ubiquinol-cytochrome c reductases or between succinate-ubiquinone and NADH-uniquinone reductases.     

Changes to the length of the flexible linker region of the Rieske protein impair the interaction of ubiquinol with the cytochrome bc1 complex.

Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c1. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol-cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent Km for ubiquinol to decrease from 9.3 to 2.6 microM. Addition of two alanine residues lowers the activity by 90% and the apparent Km decreases to 1.9 microM. Deletion of an alanine residue lowers the activity by approximately 40% and the apparent Km decreases to 5.0 microM. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol-cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc1 complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron-sulfur protein and cytochrome b.