The kinetic mechanism of ubiquinol: cytochrome c reductase at steady state.

The steady-state kinetics of ubiquinol: cytochrome c reductase (cytochrome bc1 complex) is analyzed in this work. The graphical pattern of the titrations is clearly indicative of a ping-pong mechanism, but the two products ubiquinone and reduced cytochrome c behave competitively with their substrate and noncompetitively with the other substrate. Hence, the mechanism of the reductase is of a ping-pong two-site type. A minimal reaction scheme for the enzymatic mechanism is proposed and approximate values of its rate constants are deduced on the assumption that each substrate is in rapid equilibrium at its catalytic site. This has been substantiated by presteady-state measurements of the reduction and oxidation of cytochrome b by a short-chain homolog of ubiquinol. Values of the rate constants of the reaction scheme have been deduced from the steady-state titrations for a series of 2,3-dimethoxy-5-methyl quinols having different hydrophobic substituents in position 6 of the ring. The results provide a quantitative estimation of the specificity of the quinol catalytic site in the transmembrane portion of the bc1 complex. In particular, a reasonable correlation is found between the rate of the second-order reaction of quinols with the enzyme and their solubility in lipids.     

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.     

Regulation of the proton/electron stoichiometry of mitochondrial ubiquinol:cytochrome c reductase by the membrane potential.

The electron transfer reaction catalysed by mitochondrial ubiquinol:cytochrome c reductase is linked to the outwards translocation of protons with an H+ e- stoichiometry of 1 under non-membrane potential condition. The effect of the electrical membrane potential on the H+/e- stoichiometry was investigated. The enzyme was isolated from Neurospora crassa, reconstituted into phospholipid vesicles and electrical membrane potentials of various values were generated across the membranes by means of the valinomycin-induced potassium-diffusion method. Using lithium ions as counterions for the intravesicular potassium, the induced membrane potential was stable for minutes and was not significantly changed by the protons ejected by the working enzyme. This allowed the assay of steady-state reaction rates at pre-given values of electrical membrane potential. The rate ratio between electron transfer and proton translocation declined from 1 to 0.6 with increase of the membrane potential from 0 to 100 mV. The activity of the quinol/cytochrome c redox reaction followed a parabolic dependence, being activated by low (less than 50 mV) potential and inhibited by high (greater than 100 mV) potential. This apparent non-linear dependence was interpreted in terms of a linear flow/force relationship plus a membrane-potential-dependent slip. Evaluation of the parabolic course by means of a modified linear flow/force relation also indicated a decline of the H+/e- stoichiometry from 1 to 0.5 with increase of the membrane potential from 0 to 120 mV. These observations suggest that the membrane potential controls a change of ubiquinol:cytochrome c reductase between two states that have different reaction routes.     

Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase.

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.     

Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria

The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.     

The properties of the mitochondrial succinate-cytochrome c reductase

The cytochromes b and b_T of pigeon heart mitochondria have half-reduction potentials (Em's) of +30 mV and −30 mV at pH 7.2. The midpoint potentials of these cytochromes become more negative by 30–60 mV per pH unit when the pH is made more alkaline. Detergents may be used to prepare a succinate-cytochrome c reductase free of cytochrome oxidase in which the activation of electron transport induced by oxidation of cytochrome c₁ causes the half-reduction potential of cytochrome b_T to become at least 175 mV more positive than in the absence of electron transport. This change is interpreted as indicating that the primary energy conservation reaction at site 2 remains fully functional in the purified reductase. Preliminary electron paramagnetic resonance spectra of the succinate-cytochrome c reductase as measured at near liquid helium temperatures are presented.     

Isolation and amino acid sequence of the 'Rieske' iron sulfur protein of beef heart ubiquinol:cytochrome c reductase

The sequence of the 'Rieske' iron sulfur protein from the bc1 complex of beef heart mitochondria has been determined by solid phase Edman degradation of the whole protein and of various proteolytic fragments. The protein consists of 196 amino acid residues. The molecular mass of the apoprotein was calculated to be 21,536 Da, that of the holo-protein including the Fe2S2 cluster as 21,708 Da. The protein is mainly hydrophilic with a polarity index of 42.9% and 25% of charged residues. It contains a hydrophobic membrane anchor which is predicted to form a 'hairpin' structure. The iron sulfur cluster is bound near the C-terminus of the protein between a hydrophobic and a more amphipathic domain. This reflects the fact that the cluster is located near the outer surface of the inner mitochondrial membrane. A folding pattern describing all known features of the protein is proposed.     

Isolation and amino acid sequence of the 8 kDa DCCD-binding protein of beef heart ubiquinol:cytochrome c reductase

The 8 kDa protein of beef heart ubiquinol:cytochrome c reductase was detected by means of a new SDS-PAGE [(1985) FEBS Lett. 190, 89-94] system and was isolated by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence was determined by solid-phase Edman degradation of both the N-terminal part of the whole protein and proteolytic cleavage fragments of the protein. The protein consists of 78 amino acid residues: its Mr was calculated to be 7998. Structure predictions have been made from average and sided hydropathy profiles. The suggested structure encompasses an alpha-helix and a beta-strand, the latter comprising a glutamic acid residue situated in a relatively hydrophobic neighbourhood. This residue may be responsible for the fact that the 8 kDa protein is the first subunit of the whole reductase (consisting of 11 subunits) to be labelled by DCCD when the reductase is in free form or inlaid in phospholipid vesicles.     

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria contains one cooperative ubiquinone-reduction centre

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria was isolated as a protein-Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ2) to the cytochromes c, c1 and b were studied. The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent Km value for DQH2 of the Q-free enzyme was 5 microM and of the Q-supplemented enzyme 25 microM. The pre-steady-state rate of electron transfer from DQH2 to cytochrome c1 was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre-steady-state rate of electron transfer from DQH2 to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The H+/2e- stoichiometry with DQH2 as substrate of the Q-supplemented enzyme was 3.6. These results are interpreted in accordance with a Q-cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH2-oxidation centre to the Q-reduction centre and the two monomers cooperate in the reduction of Q to QH2 at one Q-reduction centre. This centre contains two different binding sites for Q. DQH2 does not properly react at the QH2-oxidation centre. DQH2, however, binds to the loose Q-binding site of the Q-reduction centre and reduces the Q bound to the tight Q-binding site of the centre. The QH2 thus formed at the Q-reduction centre serves as electron donor for the QH2-oxidation centre.     

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.     

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.     

Enzymatic properties of ubiquinol:cytochrome c2 oxidoreductase in situ in Rhodopseudomonas palustris membranes

The presence of ubiquinol:cytochrome c2 oxidoreductase was shown in the membranes from a photosynthetic bacterium, Rhodopseudomonas palustris. Some properties of the enzyme in situ were investigated. The optimal pH of this enzyme activity was 7.0 in the intact membranes. The activity was inhibited by both antimycin and myxothiazol. Maximal activity (Vmax) was 3-4 mol cytochrome c (c2) reduced/mol cytochrome c1.s. Apparent activity of the enzyme with horse heart cytochrome c as the electron acceptor decreased as the concentration of salts in the reaction mixture increased, whereas when R. palustris cytochrome c2 was used as the electron acceptor, the activity increased as the concentration of salts increased. Moreover, the activity of the enzyme did not depend on the species or concentration of anions but on both the concentration and valency of the cations of the salts. These salt effects were thought to be due to the change of effective concentration of cytochrome molecules caused by cations near the membrane surface, which was net negatively charged. Apparent Km for ubiquinol-1 was about 80 microM irrespective of the species of cytochrome and the presence of salts.     

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.     

Comparative kinetic studies of cytochromes c in reactions with mitochondrial cytochrome c oxidase and reductase.

Kinetic studies of the reactions of selected eukaryotic and prokaryotic cytochromes c with mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase (EC 1.9.3.1) using a standardized complex IV preparation from beef heart are reported. Data on reactions with NADH-linked cytochrome c reductase (complexes I and III) are included. The concentration ranges employed provide a basis for quantitative demonstration of a general rate law applicable to oxidase reactions of cytochrome c of greatly differing reactivities. Results are interpreted on the basis of a modified Minnaert mechanism (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23), assuming productive complex formation between cytochrome c and free oxidase in addition to further complex binding of a second cytochrome c molecule to the initially formed oxidase complex. Kinetic constants so obtained are consistent with the assumption that binding is the dominant parameter in reactivity, and can be rationalized most simply on this basis.     

Dietary lipid modulation of rat liver mitochondrial succinate: cytochrome c reductase

Diets supplemented with high levels of either saturated fatty acids or unsaturated fatty acids were fed to adult rats for a period of 9 weeks and changes in the liver mitochondrial membrane phospholipid fatty acid composition and thermal behaviour of succinate: cytochrome c reductase were determined. The dietary treatment induced a change in the omega 6 to omega 3 unsaturated fatty acid ratio in the membrane lipids, with the ratio being highest with the unsaturated fatty acid and lowest with the saturated fatty acid diet. Arrhenius plots of succinate: cytochrome c reductase activity exhibited differences in both critical temperature (Tf) and Arrhenius activation energy (Ea) depending on the type of dietary treatment. The Tf was elevated from 23 degrees C in control to 32 degrees C in the saturated fatty acid-supplemented group. No significant effect on the Tf was observed in the unsaturated fatty acid-supplemented group however higher Ea values were observed due to the unsaturated fatty acid diet. The changes in succinate: cytochrome c reductase are probably due to changes in the lipid-protein interactions in the membrane, induced by the dietary lipid supplementation.     

Mechanistic differences in inhibition of ubiquinol cytochrome c reductase by the proximal Qo-site inhibitors famoxadone and methoxyacrylate stilbene

Famoxadone (FAM) is a newly commercialized antibiotic for use against plant pathogenic fungi. It inhibits mitochondria ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2, bc(1) complex) function by binding to the proximal niche of the quinol oxidation site on the enzyme. FAM has effects on the enzyme characteristic of both type Ia (E-beta-methoxyacrylates) and type Ic (stigmatellin) inhibitors. Steady-state and tight-binding inhibition kinetics; as well as direct binding measurements with famoxadone (FAM) and methoxyacrylate stilbene (MOAS), indicated that FAM is a non-competitive inhibitor of the enzyme while methoxyacrylate stilbene (MOAS) is better described as a mixed-competitive inhibitor with respect to substrate. Mixed-competitive and non-competitive inhibition kinetics predicts a ternary enzyme-substrate-inhibitor (ESI) intermediate in the reaction sequence. Current views of the Qo domain architecture propose substrate binding niches in both distal and proximal regions of the domain. Since both inhibitors bind within the proximal niche, the formation of an ESI complex implicates substrate binding within the distal niche near the iron-sulfur protein (ISP) and cytochrome c(1) (C1). In the presence of saturating FAM, addition of substrate led to a slow, nearly stoichiometric reduction of C1 that was enzyme dependent, and independent of O(2)(-) production. Similar experiments with saturating MOAS led to a slow, sub-stoichiometric reduction of C1 by substrate. A comparison of the stoichiometries of reduction, and the apparent second order rate constants (K(cat)/K(m)) indicated that saturating MOAS elicits two distinct enzyme-inhibitor (EI) intermediates. One form does not bind substrate, but the other does. In contrast, saturating FAM leads to a predominant EI form capable of binding substrate. We suggest that these differences can be correlated to the respective effects of each inhibitor on the position of the ISP, and the integrity of a distal substrate binding site. The results also indicate that binding of these inhibitory substrate analogues to the proximal niche of the Qo domain significantly increases the DeltaG(double dagger) for reduction of C1.     

X-ray diffraction by crystals of beef heart ubiquinol: cytochrome c oxidoreductase.

Beef heart mitochondrial ubiquinol:cytochrome c oxidoreductase has been crystallized in the shape of hexagonal bipyramids. At present the crystals diffract X-rays to 4.7 A. From preliminary analysis the diffraction pattern appears to be consistent with space group P6(1)22 or P6(5)22 and with unit cell parameters a = b = 212 A and c = 352 A.     

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.