A ubiquinone derivative that inhibits mitochondrial cytochrome b-c1 complex but not chloroplast cytochrome b6-f complex activity

A ubiquinone derivative, 3-chloro-5-hydroxyl-2-methyl-6-decyl- 1,4-benzoquinone (3-CHMDB), which shows different effects on the mitochondrial cytochrome b-c1 complex and chloroplast cytochrome b6-f complex, has been synthesized and characterized. When the cytochrome b-c1 complex is treated with varying concentrations of 3-CHMDB and assayed at constant substrate (Q2H2) concentration, a 50% inhibition is observed when 2 mol of 3-CHMDB per mol of enzyme are used. The degree of inhibition is dependent on the substrate concentration. When ubiquinol-cytochrome c reductase is treated with 2 mol of 3-CHMDB per mol of enzyme, less inhibition is observed with a lower substrate concentration, suggesting the possible existence of two forms of reductases: one with a high affinity for ubiquinone and another with a low affinity. 2-Chloro-5-hydroxyl-3-methyl-6-decyl-1,4-benzoquinone (2-CHMDB), an isomer of 3-CHMDB, shows much less inhibition of the mitochondrial cytochrome b-c1 complex, suggesting that the quinone binding site in this complex is highly specific. In contrast to the inhibition observed with the cytochrome b-c1 complex, 3-CHMDB causes no inhibition of the plastoquinol-plastocyanin reductase activity of chloroplast cytochrome b6-f complex, regardless of whether plastoquinol-2 or ubiquinol-2 is used as substrate. 3-CHMDB restores the dibromothymoquinone-altered EPR spectra of iron-sulfur protein in both complexes. In the case of the cytochrome b6-f complex, 3-CHMDB also partially restores the dibromothymoquinone-inhibited activity. Reduced form 3- or 2-CHMDB is oxidizable by the cytochrome b6-f complex, but not by the cytochrome b-c1 complex. These results suggest that the quinol oxidizing sites in the cytochrome b6-f complex may differ from those in the mitochondrial cytochrome b-c1 complex.     

The effects of quinone analogues on cytochrome b6 reduction and oxidation in a reconstituted system

The reconstituted system containing Photosystem I, plastocyanin and the cytochrome b6-f complex is used to study the effects of various quinone analogues on the redox behavior of cytochrome b6. The effects of DBMIB, DNP-INT and HQNO are compared in an attempt to discern the modes of action of these quinone analogues. Both DBMIB and DNP-INT are potent inhibitors of the plastocyanin reductase activity of the isolated cytochrome complex. However, while DBMIB abolished the oxidant-induced reduction of cytochrome b6, DNP-INT only inhibited about 25% of the net reduction. On the other hand, HQNO does not show any significant inhibition of plastocyanin reductase activity of the isolated cytochrome complex at concentrations up to 20 microM. An enhancement of the net amount of cytochrome b6 reduced is observed in the presence of HQNO. Both DNP-INT and HQNO inhibited the dark oxidation rate of cytochrome b6. The possible identity of the oxidant for cytochrome b6 is discussed. Plastoquinone is concluded to be the most likely candidate. DNP-INT is concluded to have at least two sites of inhibition in the cytochrome complex. The implications of these findings on quinone functions in the cytochrome b6-f complex are discussed.     

Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b6-f complex

The chloroplast cytochrome b6-f complex, incorporated into phospholipid vesicles, shows proton translocation with an observed H+/e- ratio of approximately 2. The oxidation-reduction behavior of cytochrome b6 during electron transport from duroquinol to plastocyanin is affected by incorporation. The most obvious effect of incorporation is an increase in the duration of a steady-state level of cytochrome b6 that persists during electron transport. Reagents that decrease activity increase the duration of the steady state while reagents that stimulate activity decrease this time. Uncoupling conditions yield cytochrome kinetics similar to those in the unincorporated complex. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone and 5-n-undecyl-4,7-dioxobenzothiazole inhibited reduction of cytochrome b6 in the incorporated complex, but this apparent inhibition was due to a rapid oxidation of the cytochrome by these compounds.     

DCCD binds to cytochrome b6 of a cytochrome bf complex isolated from spinach chloroplasts and inhibits proton translocation.

Radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD) was bound selectively in a time- and concentration-dependent manner to cytochrome b6 of an enzymatically active cytochrome bf complex isolated from spinach chloroplasts. Maximum labeling of cytochrome b6 was observed with 30 nmol DCCD per nmol cytochrome b6 in the cytochrome bf complex incubated for 30-60 min at 12 degrees C. After incubation of the cytochrome bf complex with DCCD under these conditions, the rate of proton ejection in the complex reconstituted into liposomes was decreased approximately 65-70% when compared to controls; however, under these same conditions the rate of electron transfer through either the soluble bf complex or the complex reconstituted into liposomes was only decreased around 20%. These results suggest that the mechanism of proton translocation through the cytochrome bf complex of spinach chloroplasts is similar to that of the cytochrome bc1 complex from yeast mitochondria in which proton pumping but not electron transfer is also inhibited by DCCD (D. S. Beattie and A. Villalobo, 1982, J. Biol. Chem. 257, 14,745-14,752).     

Binding of dicyclohexylcarbodiimide to aspartate-155 or glutamate-166 of cytochrome b6 in a cytochrome bf complex isolated from spinach thylakoids.

In a recent study [Wang & Beattie (1991) Arch. Biochem. Biophys. 291, 363-370], we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bf complex reconstituted into proteoliposomes and was bound selectively to cytochrome b6. To establish the site of binding of DCCD on cytochrome b6, the cytochrome bf complex labeled with [14C]DCCD was selectively digested with chymotrypsin and trypsin. A 17-kDa fragment containing radioactive DCCD and the heme moiety was obtained after chymotrypsin digestion, while a 12.5-kDa fragment containing both radioactive DCCD and the heme moiety was obtained after trypsin digestion, suggesting that the site of DCCD binding might be on aspartate-140, aspartate-155, or glutamate-166. Extensive digestion of cytochrome b6 isolated from a [14C]DCCD-labeled cytochrome bf complex with trypsin followed by isolation and sequencing of two radioactive peptides obtained revealed that DCCD is bound at either residue aspartate-155 or residue glutamate-166 localized in amphipathic extramembranous helix IV. In addition, the cytochrome bf complex labeled with [14C]DCCD was reconstituted into liposomes and digested with trypsin. Three fragments of 9.3, 10.5, and 11.5 kDa were obtained, suggesting that the four-helix model for the topography of cytochrome b6 in the membrane is correct.     

The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast

The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.     

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.     

DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).     

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.     

Inhibition by dicyclohexylcarbodiimide of proton ejection but not electron transfer in rat liver mitochondria

The primary effect of dicyclohexylcarbodiimide (DCCD) at the cytochrome b-c1 region of the respiratory chain of rat liver mitochondria is an inhibition of proton translocation. No significant decrease was observed in the rate of electron flow from succinate to cytochrome c when measured as cytochrome c reductase, K3Fe(CN)6 reductase, or the rate of H+ release in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone after treatment with sufficient DCCD to abolish completely electrogenic proton ejection. The inhibitory effects of DCCD were time and concentration dependent and affected by the pH of the medium. Lowering the pH from 7.3 to 6.7 resulted in a progressively faster rate and extent of inhibition of proton ejection by DCCD. At pH 6.9, the H+/2e- decreased by 50% within 30 s after DCCD addition; however, at pH 7.3, a 50% decrease was not observed until 2 min after DCCD addition. DCCD did not act as an uncoupler as both the rate of proton ejection and back decay were decreased after incubation with DCCD. Treatment of rat liver mitochondria with DCCD under these same conditions also resulted in a broadening of the sharp spectral shift of cytochrome b observed after antimycin addition to mitochondria previously reduced with succinate suggesting that DCCD may modify cytochrome b in such a way that the binding of antimycin is altered.     

NN''-dicyclohexylcarbodi-imide-sensitivity of bovine heart mitochondrial NADH: ubiquinone oxidoreductase. Inhibition of activity and binding to subunits.

Dicyclohexylcarbodi-imide (DCCD) inhibition of NADH: ubiquinone oxidoreductase was studied in submitochondrial particles and in the isolated form, together with the binding of the reagent to the enzyme. DCCD inhibited the isolated enzyme in a time- and concentration-dependent manner. Over the concentration range studied, a maximum inhibition of 85% was attained within 60 min. The time course for the binding of DCCD to the enzyme was similar to that of activity inhibition. The NADH:ubiquinone oxidoreductase activity of the submitochondrial particles was also sensitive to DCCD, and the locus of binding of the inhibitor was studied by subsequent resolution of the enzyme into subunit polypeptides. Only two subunits (molecular masses 13.7 and 21.5 kDa) were labelled by [14C]DCCD, whereas, when the enzyme in its isolated form was treated with [14C]DCCD, six subunits (13.7, 16.1, 21.5, 39, 43 and 53 kDa) were labelled. Comparison with the subunit labelling of F1F0-ATPase and ubiquinol:cytochrome c oxidoreductase indicated that the labelling pattern of NADH:ubiquinone oxidoreductase, and enzyme complex with a multitude of subunits, is unique and not due to contamination by other inner-membrane proteins. The correlation between the electron- and proton-transport functions and the DCCD-binding components remains to be established.     

Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase

The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.     

Interactions between thylakoid electron transfer complexes. II. Modification studies with glutaraldehyde

Photosystem I (PSI) and photosystem II (PSII) complexes have been isolated from stacked spinach thylakoid membranes that had been treated with varying amounts of glutaraldehyde. The concentrations of cytochrome f, Q, and P700 have been determined by spectrophotometric methods. It was found that at low concentrations of glutaraldehyde, the amount of cytochrome f associated with either PSII or PSI increased significantly while the amounts of Q and P700 stayed relatively constant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analyses indicated the presence of cytochrome f and other components of the cytochrome b6-f complex in the PSII and PSI preparations after glutaraldehyde treatment, but no intermolecular cross-linked polypeptides could be detected. Solubilization of the cytochrome b6-f complex was also inhibited after thylakoid membranes were treated with low concentrations of glutaraldehyde. These results are discussed in relation to current models for the organization of the membrane complexes, and relate to the location of the cytochrome b6-f complex in appressed and nonappressed membrane regions of thylakoids.     

Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude that Asp(B187) plays no crucial role in the protonogenic reactions of bc(1) complex, since its replacement by asparagine does not lead to any significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD.     

Structural asymmetry of the F1 of Escherichia coli as indicated by reaction with dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) inhibits the ATPase activity of F1 from Escherichia coli by covalent modification of a single glutamic acid in the beta subunit. 95% inhibition was obtained after incorporation of around 1 mole of DCCD per mole F1, i.e. 1 mole of reagent per 3 beta subunits; and up to 2 moles of DCCD per mole F1 were readily incorporated into the protein. One of the 3 beta subunits per F1 can be crosslinked to the epsilon subunit by 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC). This beta subunit (beta 1) is here shown to be shielded from reaction with DCCD, presumably by its association with epsilon and also possibly the gamma subunit. Thus the three beta subunits are not equivalent in the enzyme complex.     

Identification of a Mr = 17,000 protein as the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts

An azidoquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone (azido-Q), was used to study the plastoquinone-protein interaction and to identify the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts. When the lipid- and plastoquinone-deficient cytochrome b6-f complex is incubated with varying concentrations of azido-Q and illuminated with long wavelength UV light for 7 min at 2 degrees C, the enzymatic activity, assayed after reconstitution with lipid, decreases as the concentration of azido-Q increases. Maximum inactivation (45%) is observed when 30 mol of azido-Q is used per mol of cytochrome f. The extent of the decrease in activity upon illumination correlates with the amount of azido-Q incorporated into the protein. The 50% inactivation is in good agreement with that expected based on the amount of plastoquinone deficiency of the isolated enzyme complex. When the photolyzed, [3H]azido-Q-treated sample is extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity is found primarily in the Mr = 17,000 subunit. When the enzyme is pretreated with the electron transfer inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, significantly less radioactive label is observed in the Mr = 17,000 protein, suggesting that the action sites of these inhibitors are the same or near the plastoquinone-binding site. When the deficient complex is reconstituted with glycolipid prior to the addition of azido-Q, less than 5% inactivation is observed upon photolysis, and the amount of radioactive label on the Mr = 17,000 protein decreases greatly, suggesting that the plastoquinone-binding site is easily masked by glycolipid when endogenous plastoquinone is absent. Plastoquinol-2 apparently competes with azido-Q for the plastoquinone-binding site since it decreases the radioactive label on the Mr = 17,000 protein.     

Reconstitution of isolated Rieske Fe-S protein into a Rieske-depleted cytochrome b6-f complex

The Rieske Fe-S protein can be isolated from the cytochrome b6-f complex by means of chromatography on a hydroxyapatite column in the presence of detergent. Depletion of the cytochrome complex from the Rieske protein results in the loss of oxidoreductase activity, as well as the ability to reduce cytochrome b6. The Rieske Fe-S protein can be reconstituted into the Rieske-depleted complex by removal of the Triton X-100 molecules associated with the protein fractions, and their substitution by lipids. Upon reconstitution the complex is reactivated, and the role of the Rieske Fe-S protein in the reduction of both plastocyanin and cytochrome b6 can be demonstrated.     

Effect of dicyclohexylcarbodiimide on unisite and multisite catalytic activities of the adenosinetriphosphatase of Escherichia coli

The inhibitory effect of dicyclohexylcarbodiimide (DCCD) on the activity of the adenosine-triphosphatase of Escherichia coli (ECF1) has been examined in detail. DCCD reacted with ECF1 predominantly in beta subunits with a maximum of 2 mol of reagent per mole of ECF1 being incorporated in these subunits. Ninety-five percent inhibition of steady-state or multistate ATPase activity required incorporation of 1 mol of DCCD per mole of enzyme into beta subunits. Seventy-five percent inhibition of the initial rate of unisite catalysis was only obtained after incorporation of 2 mol of DCCD per mole of ECF1 into beta subunits. Analyses of the kinetics of unisite catalysis and nucleotide binding experiments both indicate that DCCD binds outside the substrate ATP binding site. Inhibition by this reagent appears to be due in part to an effect on the catalytic sites but mainly to the blocking of cooperativity between these sites.     

Dicyclohexylcarbodiimide binds to cytochrome b and subunit VIII in soluble complex III from beef heart mitochondria

Incubation of soluble complex III isolated from either yeast or beef heart mitochondria with 25-100 nmol of [14C]dicyclohexylcarbodiimide (DCCD)/nmol of cytochrome b followed by centrifugation through 10% sucrose or precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunits of either complex. The [14C]DCCD was bound to cytochrome b and phospholipids in the yeast complex and with similar kinetics to both cytochrome b and subunit VIII (Mr = 4000-8000) plus phospholipids of the beef complex. Subunit VIII of the beef complex was partially extracted with chloroform:methanol; however, no subunit of this mobility was present in the yeast complex. Incubation of the beef complex in phosphate buffer for short times resulted in a doubling of the [14C]DCCD bound to cytochrome b relative to that to subunit VIII. Preincubation of both complexes with venturicidin prior to treatment with DCCD resulted in a 50% decrease in the binding of [14C]DCCD to cytochrome b. Reisolation of the beef complex III by precipitation with (NH4)2SO4 after incubation with [14C]DCCD resulted in the formation of a new band with an apparent molecular weight of 39,000 even in the zero time control. The [14C]DCCD was bound to subunit VIII and the core proteins but not to cytochrome b at all times, suggesting that precipitation with (NH)2SO4 in the presence of DCCD causes cross-linking of the subunits of complex III.     

Chemical modification of the mitochondrial bc1 complex by N,N'-dicyclohexylcarbodiimide inhibits proton translocation

We report here that N,N'-dicyclohexylcarbodiimide (DCCD) decreases the H/2e stoichiometry of the cytochrome bc1 complex from 3.8 +/- 0.2 (10) to 2.1 +/- 0.1 (8) but has only a minimal effect on the H/2e ratio of cytochrome oxidase under the relatively mild conditions used. The effect on the bc1 complex cannot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage. We conclude that DCCD is an inhibitor of proton translocation within the bc1 complex. There are three possible explanations of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reactions but not electron transport, (c) DCCD could prevent the conduction of the translocated proton to the external phase.