Low-resolution structural studies of mitochondrial ubiquinol:cytochrome c reductase in detergent solutions by neutron scattering

Mitochondrial ubiquinol:cytochrome c reductase (Mr approximately 600,000) was cleaved into a complex (Mr approximately 280,000) of the subunits III (cytochrome b), IV (cytochrome c1) and VI to IX, a complex (Mr approximately 300,000) of the subunits I and II, and the single subunit V (iron-sulphur subunit, Mr approximately 25,000). Neutron scattering was applied to the whole enzyme, the cytochrome bc1 complex, both in hydrogenated and deuterated alkyl (phenyl) polyoxyethylene detergents, and the complex of subunits I and II in detergent-free solution. The neutron parameters were compared with the structures of the enzyme and the cytochrome bc1 complex previously determined by electron microscopy. Using the method of hard spheres, comparison of the calculated and experimental radius of gyration implies that the length of the enzyme across the bilayer or the detergent micelle is between 150 and 175 A and of the cytochrome bc1 complex between 90 and 115 A. The subunit topography was confirmed. The cleavage plane between the cytochrome bc1 complex and the complex of subunits I and II lies at the centre of the enzyme and runs parallel to the membrane just outside the bilayer. The detergent uniformly surrounds the protein as a belt, which is displaced by 30 to 40 A from the protein centre of the enzyme and by about 20 A from the protein centre of the cytochrome bc1 complex. The low protein matchpoint of the whole enzyme as compared to the subunit complexes is accounted for in terms of the non-exchange of about 30 to 60% of the exchangeable protons within the intact enzyme. Polar residues are, on average, at the protein surface and non-polar residues and polar residues with non-exchanged protons are buried within the enzyme.     

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.     

Membrane-bound and water-soluble cytochrome c1 from Neurospora mitochondria

Cytochrome c1 is a subunit of ubiquinol--cytochrome c reductase (EC 1.10.2.2). In Neurospora crassa wild type 74A grown in the presence of chloramphenicol, the subunit is inserted only into the bilayer of the mitochondrial inner membranes without associating with other proteins. From these modified membranes a monodisperse (cytochrome c1)-Triton complex was isolated by subjecting the Triton-solubilized membranes to affinity chromatography on immobilized cytochrome c. A water-soluble pentamer of cytochrome c1 was prepared from the (cytochrome c1)-Triton complex by removing the detergent. By limited proteolytic digestion of the cytochrome c1-Triton complex with chymotrypsin, a water-soluble monomeric cytochrome c1 was prepared which has a molecular weight of only 24 000 as compared to 31 000 of the membrane-bound cytochrome c1. The 24 000-Mr cytochrome c1 and the 31 000-Mr cytochrome c1 have same light absorption spectra and cytochrome-c-binding properties. These results are used to propose the following model. Cytochrome c1 consists of a large hydrophilic part and a small hydrophobic part. The hydrophilic part extends from the mitochondrial inner membrane into the intermembrane space. This part carries the heme and interacts with cytochrome c. The hydrophobic part anchors the cytochrome c1 to the bilayer.     

Purification of a reconstitutively active iron-sulfur protein (oxidation factor) from succinate . cytochrome c reductase complex of bovine heart mitochondria.

Oxidation factor, a protein required for electron transfer from succinate to cytochrome c in the mitochondrial respiratory chain, has been purified from isolated succinate . cytochrome c reductase complex. Purification of the protein has been followed by a reconstitution assay in which restoration of ubiquinol . cytochrome c reductase activity is proportional to the amount of oxidation factor added back to depleted reductase complex. The purified protein is a homogeneous polypeptide on acrylamide gel electrophoresis in sodium dodecyl sulfate and migrates with an apparent Mr = 24,500. Purified oxidation factor restores succinate . cytochrome c reductase and ubiquinol . cytochrome c reductase activities to depleted reductase complex. It is not required for succinate dehydrogenase nor for succinate . ubiquinone reductase activities of the reconstituted reductase complex. Oxidation factor co-electrophoreses with the iron-sulfur protein polypeptide of ubiquinol . cytochrome c reductase complex. The purified protein contains 56 nmol of nonheme iron and 36 nmol of acid-labile sulfide/mg of protein and possesses an EPR spectrum with the characteristic "g = 1.90" signal identical to that of the iron-sulfur protein of the cytochrome b . c1 complex. In addition, the optimal conditions for extraction of oxidation factor, including reduction with hydrosulfite and treatment of the b . c1 complex with antimycin, are identical to those which facilitate extraction of the iron-sulfur protein from the b . c1 complex. These results indicate that oxidation factor is a reconstitutively active form of the iron-sulfur protein of the cytochrome b . c1 complex first discovered by Rieske and co-workers (Rieske, J.S., Maclennan, D.H., and Coleman, R. (1964) Biochem. Biophys. Res. Commun. 15, 338-344) and thus demonstrate that this iron-sulfur protein is required for electron transfer from ubiquinol to cytochrome c in the mitochondrial respiratory chain.     

Reconstitution of the ubiquinol: cytochrome c reductase from a bc1 subcomplex and the 'Rieske' iron-sulfur protein isolated by a new method

1. A method for preparing the 'Rieske' iron-sulfur protein and the bc1 subcomplex of complex III was developed. The new method is advantageous over the published ones in that: (a) the final yield and amount exceeds by far those obtained when employing the hitherto published methods; (b) the iron-sulfur protein as well as the bc1 subcomplex are obtained by one and the same preparation procedure from a common source; and (c) the preparation method is easier than the published ones. 2. The iron-sulfur protein obtained represents the first reconstitutively active preparation present in a monodisperse state. 3. The reconstitution of the ubiquinol:cytochrome c reductase from the two components is a reversible dissociation process. Full activity of ubiquinol:cytochrome c reductase is reached after saturation of the binding site of the bc1 subcomplex for iron-sulfur protein. 4. Full reduction of the constituent cytochrome c1 of the bc1 subcomplex can already be obtained with substoichiometric amounts of iron-sulfur protein, however. 5. The question might be raised whether the observed dissociation equilibrium represents merely a phenomenon occurring specifically with the proteins isolated in Triton X-100 and investigated in a Triton-containing buffer, or whether dissociation of the iron-sulfur protein also takes place in the mitochondrial membrane in the course of the electron-transfer reaction sequence.     

Electron transfer in monomeric forms of beef and shark heart cytochrome c oxidase

Beef heart cytochrome c oxidase is dimeric in reconstituted membranes and in nonionic detergents at physiological pH [Henderson, R., Capaldi, R. A., & Leigh, J. (1977) J. Mol. Biol. 112, 631; Robinson, N.C., & Capaldi, R. A. (1977) Biochemistry 16, 375], raising the possibility that this aggregation state is a prerequisite for enzymatic activity. A procedure for dissociating the enzyme into monomers is presented. This involves treating the protein with high concentrations of Triton X-100 at pH 8.5. The electron transfer activity of the monomer is comparable to that of the dimer under identical assay conditions. The beef heart cytochrome c oxidase monomer was found to be heterogeneous in hydrodynamic studies, probably due to dissociation of associated polypeptides, including subunit III. Monomer molecular weights in the range 129 000-160 000 were obtained. Previous studies have indicated that shark heart cytochrome c oxidase is monomeric under physiological conditions. Sedimentation equilibrium studies reported here confirm this. The elasmobranch enzyme, with a similar polypeptide composition to that of beef enzyme, was determined to have a molecular weight of 158 000.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretically monodisperse cytochrome c oxidases

A discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions has been used to demonstrate monodispersity of procaryotic and eucaryotic cytochrome c oxidase preparations. Alkaline treated bovine enzyme which contains nine subunits as analysed by subsequent discontinuous SDS-polyacrylamide gel electrophoresis is a monodisperse dimer in 0.1% Triton X-100 and a monomer in 0.1% dodecyl maltoside. The Mr-values corrected for bound detergent are 286,000 in Triton X-100 and 152,000 in dodecyl maltoside respectively. The two-subunit bacterial cytochrome c oxidase of Paracoccus denitrificans is proved to be a monomer with a corrected Mr of 76,000 in both nonionic detergents Triton X-100 and dodecyl maltoside.     

Purification and characterization of cytochrome c oxidase from rat liver mitochondria.

Cytochrome c oxidase has been purified from rat liver mitochondria using affinity chromatography. The preparation contains 10.5 to 13.4 nmol of heme a + a3 per mg of protein and migrates as a single band during polyacrylamide gel electrophoresis under nondissociating conditions. It has a heme a/a3 ratio of 1.12 and is free of cytochromes b, c, and c1 as well as the enzymes, NADH dehydrogenase, succinic dehydrogenase, coenzyme Q-cytochrome c reductase, and ATPase. The enzyme preparation consists of six polypeptides having apparent Mr of 66,000, 39,000, 23,000, 14,000, 12,500 and 10,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide composition is similar to those found for cytochrome c oxidases from other systems. The enzymatic activity of the purified enzyme is completely inhibited by carbon monoxide or cyanide, partially inhibited by Triton X-100 and dramatically enhanced by Tween 80 or phospholipids.     

Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri.

Nitric oxide (NO) reductase was solubilized by Triton X-100 from the membrane fraction of Pseudomonas stutzeri ZoBell and purified 100-fold to apparent electrophoretic homogeneity. The enzyme consisted of two polypeptides of Mr 38,000 and 17,000 associated with heme b and heme c, respectively. Absorption maxima of the reduced complex were at 420.5, 522.5, and 552.5 nm, with a shoulder at 560 nm. The electron paramagnetic resonance spectrum was characteristic of high- and low-spin ferric heme proteins; no signals typical for iron-sulfur proteins were found. Nitric oxide reductase stoichiometrically transformed NO to nitrous oxide in an ascorbate-phenazine methosulfate-dependent reaction with a specific activity of 11.8 mumols/min per mg of protein. The activity increased to 40 mumols upon the addition of soybean phospholipids, n-octyl-beta-D-glucopyranoside, or its thio derivative to the assay system. Apparent Km values for NO and phenazine methosulfate were 60 and 2 microM, respectively. The pH optimum of the reaction was at 4.8. Cytochrome co was purified from P. stutzeri to permit its distinction from NO reductase. Spectrophotometric binding assays and other criteria also differentiated NO reductase from the respiratory cytochrome bc1 complex.     

Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).

NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.     

Functional activities of monomeric and dimeric forms of the chloroplast cytochrome b6f complex

A monomeric form of the isolated cytochrome b6f complex from spinach chloroplast membranes has been isolated after treatment of the dimeric complex with varying concentrations of Triton X-100. The two forms of the complex are similar as regards electron transfer components and subunit composition. In contrast to a previous report (Huang et al. (1994) Biochemistry 33: 4401–4409) both the monomer and dimer are enzymatically active. However, after incorporation of the respective complexes into phospholipid vesicles, only the dimeric form of the cytochrome complex shows uncoupler sensitive electron transport, an indication of coupling of electron transport to proton translocation. The absence of this activity with the monomeric form of the cytochrome complex may be related to an inhibition by added lipids.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - mega-9- nonanoyl-N-methylglucamide     

Three-dimensional structure of NADH: ubiquinone reductase (complex I) from Neurospora mitochondria determined by electron microscopy of membrane crystals

NADH: ubiquinone reductase (electron transfer complex I) has been isolated from Neurospora crassa mitochondria as a monodisperse protein-phospholipid-Triton X-100 complex (1:0.04:0.15, by weight). The enzyme is in the monomeric state, has a protein molecular weight of 610,000 and consists of about 25 different subunits. Membrane crystals of the enzyme complex have been prepared by adding mixed phospholipid-Triton X-100 micelles and then removing the Triton by dialysis. Diffraction patterns of the negatively stained membrane crystals extend to about 3.9 nm, with a unit cell size of 19 nm X 38 nm and gamma = 90 degrees. The two-sided plane group packing corresponding to pgg is p22(1)2(1). By combining four sets of tilted views, a low-resolution three-dimensional structure of the protein has been calculated. The structure shows that NADH: ubiquinone reductase extends 15 nm across the membrane, projecting 9 nm from one membrane side and 1 nm from the opposite side. Only about one-third of the total protein mass is located in the membrane. The structure of NADH: ubiquinone reductase is compared with that of ubiquinol: cytochrome c reductase determined by electron microscopy of membrane crystals.     

Characterisation of binding of the methoxyacrylate inhibitors to mitochondrial cytochrome c reductase

The three E-beta-methoxyacrylate (MOA) inhibitors oudemansin A, strobilurin A and MOA stilbene [3-methoxy-2(2-styrylphenyl)propenic acid-methylester], which differ by more than one order of magnitude in their binding affinity to the mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc1 complex), bind to a site that is not identical to the binding site for ubihydroquinone, the substrate of the outer ubiquinone reaction site (Qo centre). Although the ubihydroquinone molecule is still bound in the presence of the MOA inhibitors, its electrons cannot be transferred to the iron-sulfur centre. A shift of the relative position of the ubihydroquinone molecule in the reaction centre due to a conformational distortion of cytochrome b induced by the binding of the MOA inhibitor seems to be the reason for the blocked electron transfer. Further analysis shows that ubihydroquinone affects the Kd values of all three MOA inhibitors tested: the values are raised by a constant factor of two, although the inhibitors bind with quite different affinity. The iron-sulfur protein is not involved in the binding of the MOA inhibitors. These results have direct implications for the proper use of MOA inhibitors in experiments designed to analyse the structure/mechanism relationship in cytochrome c reductase. In particular, point mutations recently described in MOA-inhibitor-resistant mutants can no longer be taken to affect necessarily the ubihydroquinone binding site.     

Purification and some characteristics of a cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes

Nitrous oxide reductase from Wolinella succinogenes was purified very nearly to homogeneity. The enzyme was found to be dimeric, with Mr = 162,000 and subunit Mr = 88,000, and to contain three copper atoms and one iron atom (as cytochrome c) per subunit. The oxidized enzyme exhibited absorption bands at 410 and 528 nm, and the dithionite-reduced enzyme, at 416, 520, and 550 nm. The isoelectric point was 8.6; specific activity was at 25 degrees C and pH 7.1, 160 mumol x min-1 x mg-1; and Km was 7.5 microM N2O under the same conditions. alpha-Chymotrypsin cleaved the enzyme into cytochrome c-depleted dimers with an average Mr = 134,000 and cytochrome c-enriched fragments with an average Mr = 13,000. The enzyme was stable at 4 degrees C for at least 100 h under air and 3 h in the presence of 5 mM EDTA. It exhibited a dithionite-N2O oxidoreductase as well as a BV+-N2O oxidoreductase activity. During turnover with BV+ at 25 mM N2O, the enzyme was observed to undergo an initial activation and a subsequent inactivation. The kinetics of inactivation were approximately first-order in remaining activity, and the first-order rate constant was essentially independent of the initial enzyme concentration. These characteristics are consistent with the occurrence of turnover-dependent inactivation. Acetylene was a relatively weak inhibitor, but cyanide and azide were rather strong inhibitors. The nitrous oxide reductase of W. succinogenes is quite different from that of denitrifying bacteria. The amount of activity in cell extracts and the absence of O2-labile nitrous oxide reductase suggested that the cytochrome c containing enzyme may be the only one produced by W. succinogenes.     

Separation of enzymically active bovine cytochrome c oxidase monomers and dimers by high performance liquid chromatography

The aggregation state of two types of bovine heart cytochrome c oxidase preparations in the presence of laurylmaltoside was investigated by high performance liquid chromatography in two buffers of ionic strengths of 388 mM and 45 mM, respectively. At high ionic strength, it was found that the Fowler cytochrome c oxidase preparation was monomeric (Mr = 2 X 10(5)), while monomers and dimers (2 X aa3, Mr = 4 X 10(5)) could be isolated from the Yonetani preparation. Under these conditions there was no rapid equilibrium between the two forms. Covalent cytochrome c oxidase-cytochrome c complexes were largely dimeric, and addition of ascorbate and cytochrome c to the oxidase also promoted dimerization. At low ionic strength (I = 45 mM) in the presence of laurylmaltoside the oxidase and the covalent complex with cytochrome c were largely monomeric. In the steady-state oxidation of ferrous horse heart cytochrome c, the monomeric enzyme displayed biphasic kinetics at I = 45 mM. This suggests that the presence of high- and low-affinity reactions is an intrinsic property of the cytochrome c oxidase monomer.     

An active cytochrome c oxidase depleted of subunit III prepared by covalent chromatography on yeast cytochrome c

A covalent chromatography technique is described for the preparation of an active cytochrome c oxidase from bovine heart devoid of subunit III. Yeast cytochrome c is immobilized on a Sepharose 4B gel, its cysteine 107 activated and reacted with the oxidase. Elution with Triton X-100 releases an oxidase devoid of subunit III, which is recovered after elution with β-mercaptoethanol.     

Electron microscopic characterization of helical filaments formed by subunits I and II (core proteins) of ubiquinol: cytochrome c reductase from Neurospora mitochondria

The isolated and water-soluble complex of subunits I and II (core proteins) of ubiquinone:cytochrome c reductase from Neurospora mitochondria forms filaments below pH 6.0. Three independent helical reconstructions of single filaments were compared with the 3-D reconstruction of the native enzyme. A model for the helix is proposed in which the core complex dimers are arranged radially with the face which is proximal to the membrane in the native enzyme on the outside of the helix. The dimension of the core complex dimer perpendicular to the helix axis (70 A) provides an independent estimate of the height of the core complex to that obtained previously from cytochrome reductase crystals. The results of STEM mass measurement and the helical model give a mass per repeating unit of 90 kDa, which would indicate that the monomeric core complex consists of one 45-kDa and one 50-kDa subunit.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.     

Affinity purification of cytochrome c reductase from potato mitochondria.

Ubiquinol-cytochrome-c oxidoreductase has been isolated from potato (Solanum tuberosum L.) mitochondria by cytochrome-c affinity chromatography and gel-filtration chromatography. The procedure, which up to now only proved applicable to Neurospora, yields a highly pure and active protein complex in monodisperse state. The molecular mass of the purified complex is about 650 kDa, indicating that potato cytochrome c reductase occurs as a dimer. Upon reconstitution into phospholipid membranes, the dimeric enzyme catalyzes electron transfer from a synthetic ubiquinol to equine cytochrome c with a turnover number of 50 s-1. The activity is inhibited by antimycin A and myxothiazol. A myxothiazol-insensitive and antimycin-sensitive transhydrogenation reaction, with a turnover number of 16 s-1, can be demonstrated as well. The protein complex consists of ten subunits, most of which have molecular masses similar to those of the nine-subunit fungal enzyme. Individual subunits were identified immunologically and spectral properties of b and c cytochromes were monitored. Interestingly, an additional 'core' polypeptide which is not present in other cytochrome bc1 complexes forms part of the enzyme from potato. Antibodies raised against individual polypeptides reveal that the core proteins are clearly immuno-distinguishable. The additional subunit may perform a specific function and contribute to the high molecular mass which exceeds those reported for other cytochrome-c-reductase dimers.