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.     

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.     

Time and concentration dependence of the dicyclohexylcarbodiimide inhibition of proton movements in the cytochromebc 1 complex from yeast mitochondria reconstituted into proteoliposomes

The cytochromebc ₁ complex was isolated from yeast mitochondria solubilized with the detergent dodecyl maltoside and reconstituted into proteoliposomes to measure electrogenic proton pumping. Optimal respiratory control ratios of 4.0, obtained after addition of the uncoupler CCCP, and H⁺/e ^– ratios of 1.6 were obtained when the proteoliposomes were prepared with egg yolk phosphatidylcholine supplemented with cardiolipin. Moreover, it was critical to remove excess dodecyl maltoside in the final concentrated preparation prior to reconstitution to prevent loss of enzymatic activity. The rate of electrogenic proton pumping, the respiratory control ratios, and the H⁺/e ^– ratios were decreased by incubation of the cytochromebc ₁ complex with dicyclohexylcarbodiimide (DCCD) in a time and concentration dependent manner. Maximum inhibitions were observed when 50 nmol DCCD per nmol of cytochromeb were incubated for 30 min at 12°C with the intact cytochromebc ₁ complex. Under these same conditions maximum labeling of cytochromeb with [¹⁴C] DCCD was reported in a previous study [Beattieet al. (1984).J. Biol. Chem. 259, 10562–10532] consistent with a role for cytochromeb in electrogenic proton movements.     

A proposed pathway of proton translocation through thebc complexes of mitochondria and chloroplasts

The cytochromebc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50–70% with minimal effect on electron transfer in the cytochromebc ₁ and cytochromebf complexes reconstituted into liposomes. Subsequent binding studies with cytochromebc ₁ and cytochromebf complexes indicate that DCCD specifically binds to the subunitb and subunitb ₆, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochromeb and aspartate 155 or glutamate 166 of spinach cytochromeb ₆. Moreover, similar inhibition of proton translocating activity and binding to cytochromeb and cytochromeb ₆ were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochromeb and cytochromeb ₆ is localized near the surface of the membrane but shielded from the external medium.     

Effect of ubiquinone extraction on the reaction of the mitochondrialbc 1 complex with ferricyanide

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochromeb extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochromec was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/molbc ₁ complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochromeb extrareduction. Since the initial rates of cytochromeb reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochromeb was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.     

Evidence for a Conformational Change in Subunit III of Bovine Heart Mitochondrial Cytochrome c Oxidase1

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl—biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.     

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.     

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.     

Antimycin-resistant alternate electron pathway to plastocyanin in bovine-heart Complex III

Bovine-heart Complex III can catalyze the reduction of spinach plastocyanin by a decyl analog of ubiquinol-2 at a rate comparable with the rate of plastocyanin reduction by plastoquinol as catalyzed by the cytochromeb ₆–f complex purified from spinach leaves. This plastocyanin reduction as catalyzed by Complex III was almost completely inhibited by myxothiazol at stoichiometric concentrations, partially inhibited by UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) and funiculosin, and was relatively insensitive to antimycin and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). Cytochromec reduction as catalyzed by Complex III displayed a residual, inhibitor-insensitive rate of 5% of the uninhibited rate for each of the three inhibitors, antimycin, myxothiazol, and UHDBT. However, the residual rate that was insensitive to each of the inhibitors added singly was inhibited further by addition of the remaining two inhibitors. From these results it is concluded that plastocyanin reduction involves an electron-transfer pathway through Complex III that is distinct from the pathway utilized for reduction of cytochromec.A portion of the data in this report was presented at the IV International Symposium on Coenzyme Q₁₀, Munich, F.R.G., November, 6–9, 1983.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

Orientation of complex III in the yeast mitochondrial membrane: Labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [¹²⁵I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochromeb revealed that the two core proteins and cytochromeb were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochromec ₁ and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromesb andc ₁ was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15–30% of the total dithionite-reducibleb and 100% of the cytochromec ₁: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochromeb but insignificant amounts of cytochromec ₁. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.Abbreviations used: DABS, diazobenzene sulfonate; DBH₂, reduced form of decyl analogue of coenzyme Q (2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.     

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase to inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II–IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site in subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.     

A durohydroquinone oxidation site in the mitochondrial transport chain

Although duroquinone had little effect upon NADH oxidation in neutral lipid depleted mitochondria, durohydroquinone was oxidized by ETP at a rate sensitive to antimycin A. Fractionation of mitochondria into purified enzyme systems showed durohydroquinone: cytochromec reductase to be concentrated in NADH: cytochromec reductase, absent in succinate:cytochromec reductase, and decreased in reduced coenzyme Q:cytochromec reductase. Durohydroquinone oxidation could be restored by recombining reduced coenzyme Q:cytochromec reductase with NADH:coenzyme Q reductase. Pentane extraction had no effect upon either durohydroquinone or reduced coenzyme Q₁₀ oxidation, indicating lack of a quinone requirement between cytochromesb andc. Both chloroquine diphosphate and acetone (96%) treatment irreversibly inhibited NADH but not succinate oxidation. Neither reagents had any effect upon durohydroquinone oxidation but both inhibited reduced coenzyme Q₁₀ oxidation 50%, indicating a site of action between Q₁₀ and duroquinone sites. Loss of chloroquine sensitive reduced coenzyme Q₁₀ oxidation after acetone extraction suggests two sites for Q₁₀ before cytochromeb.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

Isolation of totally inverted submitochondrial particles by sonication of beef heart mitochondria

A novel procedure for isolating totally inverted preparations of submitochondrial particles by sonication of beef heart mitochondria is described. The procedure involves only differential centrifugation in 0.25 M sucrose containing 0.15 M KCl. The submitochondrial particles have 96% of their cytoplasmic face cytochromec-binding sites sequestered within the particles. Mild sonication exposes cytochromec-binding sites to the medium. The oligomycin-sensitive ATPase of sonic-derived submitochondrial particles, like that of electron transport particles, is inhibited 98% by exogenous isolated ATPase inhibitor protein. NADH oxidase activity in these particles is inhibited by oligomycin. The respiratory control index (uncoupled rate/oligomycin-inhibited rate) is approximately 3.4 and can be increased by washing the particles with medium containing bovine serum albumin.     

Effect of trypsin on the kinetic properties of reconstituted beef heart cytochromec oxidase

Isolated beef heart cytochromec oxidase was reconstituted in liposomes by the cholate dialysis method with 85% of the binding site for cytochromec oriented to the outside. Trypsin cleaved specifically subunit VIa and half of subunit IV from the reconstituted enzyme. The kinetic properties of the reconstituted enzyme were changed by trypsin treatment if measured by the spectrophotometric assay but not by the polarographic assay. It is concluded that subunit VIa and/or subunit IV participate in the electron transport activity of cytochromec oxidase.     

The effect of membrane potential on the redox state of cytochromeb 561 in antimycin-inhibited submitochondrial particles

The oxidation of cytochromeb ₅₆₁ by ATP was measured in submitochondrial particles inhibited by antimycin. The redox potential of the bulk (M phase) was controlled by the ratio of fumarate:succinate, and the oxidation of cytochromeb was calculated and expressed as a change in redox potential (E _h) measured in millivolts. The oxidation of cytochromeb ₅₆₁ is an energy-driven reaction affected only by the component of the proton motive force. The oxidation (measured in millivolts) is a function of the phosphate potential, reaching a maximal value of 40 mV at G_ATP<–12 kcal/mole. The maximal measured value of ATP-dependent was 100 mV. Thus only a fraction of the membrane potential effects the redox state of cytochromeb ₅₆₁. In contrast to the ATP-induced oxidation of cytochromeb ₅₆₁, cytochromeb ₅₆₆ is in redox equilibrium with fumarate succinate either in the presence or in the absence of ATP. The selective oxidation ofb ₅₆₁ is explained within the term of theQ cycle as a reflection of on the electron electrochemical potential. The positive electric potential of theC phase causes cytochromeb ₅₆₆ to act as oxidant with respect to cytochromeb ₅₆₁. In the presence of antimycin cytochromeb ₅₆₁ cannot equilibrate with the quinone and undergoes oxidation, while cytochromeb ₅₆₆ reequilibrates with the quinone and thus regains redox equilibrium with the fumarate succinate redox buffer.Abbreviations used: ETP_H, phosphorylating submitochondrial particles; TMPD,N ₁ N ₁ NN-tetramethyl-p-phenylenediamine; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydrazone; Mes, 2-(N-morpholino) ethanesulfonic acid.     

Structural aspects of the cytochromeb 6 f complex; structure of the lumen-side domain of cytochromef

The following findings concerning the structure of the cytochromeb ₆ f complex and its component polypeptides, cytb ₆, subunit IV and cytochromef subunit are discussed: