The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

Studies on the orientations of the mitochondrial redox carriers. Orientation of the chromophores of cytochrome b--c1 complex with respect to the plane of a cytochrome b--c1 complex--lipid model membrane.

Orientations of the active site chromophores of the mitochondrial cytochrome b-c₁ complex incorporated into liposomes have been investigated in hydrated oriented multilayers of proteoliposome membranes using optical and epr spectroscopy. The hemes of cytochromes c₁ and b were found to be oriented with the normal to their heme planes lying approximately in the plane of the proteoliposome membrane. Rieske's iron-sulfur center was oriented with the z-axis of the g tensor parallel to the plane of the membranes. It is concluded that the cytochrome b-c₁ complex has a structural asymmetry which causes it to orient with respect to the lipid bilayer.     

Optical and Potentiometric Study of the b- and c-Type Cytochromes in Mushroom Agaricus bisporus Lge Mitochondria

Differential spectrometry revealed two species for the b-type, as well as for the c-type, cytochromes in mitochondria from Agaricus bisporus Lge. The two b-type components are denoted according to their peak position in the α region at room temperature, i.e. b₅₆₀ and b₅₆₆. The b₅₅₆ component present in all the studied higher plant mitochondria was not detected in the system. At 293 K, the c-type cytochromes exhibit a common α band with a maximum at 550 nanometers. This band is split at 77 K, with peak positions at 547 nanometers (cytochrome c) and 552 nanometers (cytochrome c₁).     

Properties of Ascaris muscle mitochondria. I. Cytochromes

1. The cytochrome system in Ascaris muscle mitochondria was further characterized using purer preparations.2. Difference spectra (at 22 °C and ?196 °C) of the mitochondrial preparations using succinate and ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine show that Ascaris muscle mitochondria contain cytochromes c₁, c and aa₃, and also at least three b-type cytochromes. The b-type cytochrome is the predominant component.3. Cytochrome c and Ascaris cytochrome b-560 can be extracted from the mitochondrial preparations with 150 mM KCl, leaving the membrane-bound cytochromes c₁, b and aa₃ in the KCl residue.     

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles

A kinetic study on ubiquinol-cytochrome f reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c₁ complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a K_m for ubiquinol-1 of 13.4 μM in mitochondria and of 24.6 μM in the isolated cytochrome b-c₁ complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent K_m values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the V_max and the K_m for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.     

The Cytochromes of Prototheca zopfii

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a₃. Cytochromes a and a₃ could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a₃ complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a₃, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).     

Development and endocrine regulation of mitochondrial cytochrome biosynthesis in the insect fat body: δ-[14C]aminolevulinic acid incorporation

The rate of incorporation of [¹⁴C]aminolevulinic acid (ALA) into cytochrome hemes was used to measure mitochondrial cytochrome synthesis in the fat body of adult male Blaberus discoidalis cockroaches. The hemes of cytochromes aa₃+b and c+c₁, were chemically separated to observe differential rates in their synthesis and regulation. [¹⁴C]ALA was linearly incorporated into cytochrome hemes for at least 8 h. No significant pool of endogenous ALA was detected relative to the amount of administered [¹⁴C]ALA. Peak cytochrome synthesis occurred 4 to 6 days after adult emergence. Endocrine disruption by corpora cardiaca-corpora allata extirpation or cervical ligation eliminated the 4-day developmentally related increase in the rate of cytochrome aa₃+b synthesis but had no effect on the production of cytochromes c+c₁. Injections of corpora cardiaca extracts into cervically ligated animals stimulated the rate of production of cytochromes aa₃+b by 2.5 times but did not affect cytochromes c+c₁. By comparison, juvenile hormone injections did not affect the rate of synthesis of either cytochrome fraction. These findings indicate that a neurohormone regulates the rate of synthesis of cytochromes a+b in insect fat body mitochondria.     

The use of photoactivated hetero-bifunctional reagents to cross-link cytochrome c to proteins that bind it by electrostatic interactions.

Lysine residues of horse heart cytochrome c have been modified with N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) and ethyl N-5-azido-2-nitrobenzoylaminoacetimidate (ANB-AI), reagents that attach nitroaryl azides onto the surface of proteins by amide and amidine linkages, respectively. When acting as an electron acceptor for yeast cytochrome b₂, modification of cytochrome c with ANB-NOS increases the K_m for the reaction by 2-fold, while modification with ANB-AI has little effect on the K_m. The V_max for the reduction of cytochrome c by cytochrome b₂ is reduced by the attachment of both compounds to cytochrome c. When the modified cytochromes c were illuminated with phosvitin, cytochrome b₅, and cytochrome c peroxidase, cross-linked species were formed which could be resolved by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. In each case the amidine derivatives of cytochrome c modified with ANB-AI showed more cross-linking than the amide derivatives of cytochrome c modified with ANB-NOS. When the modified cytochromes c were present in a 3-fold excess of phosvitin, cross-linked products containing 1, 2, and 3 molecules of cytochrome c covalently attached to phosvitin were observed. Photolysis of the modified cytochromes c in the presence of cytochrome b₅, resulted in the formation of a cross-linked 1:1 complex between the two cytochromes as well as higher order aggregates containing up to 5 molecules of cytochrome c plus cytochrome b₂. When cytochrome c peroxidase was illuminated with the modified cytochromes c, the predominant cross-linked product was a 1:1 complex between the two heme proteins. However, a cross-linked species was detected in small amounts with the apparent composition of 2 molecules of cytochrome c and 1 of the peroxidase. Also, a procedure is described for the synthesis of ANB-AI with ¹⁴C in the imidocarbon which is ultimately derived from ¹⁴CN.     

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.     

Mitochondrial cytochrome b-c complex: its oxidation-reduction components and their stoichiometry.

A cytochrome b-c₁ complex was isolated from pigeon breast muscle mitochondria and purified to a content of 3 nmol of cytochrome c₁ per milligram of protein. Anaerobic suspensions of the preparation were titrated with reducing equivalents (NADH) and oxidizing equivalents (ferricyanide). The oxidation-reduction components of the complex were measured by the number of reducing equivalents accepted or donated per cytochrome c₁ and compared with the stoichiometries of the known redox components as measured by independent methods. The preparation accepts or donates 5.2 ± 0.3 equivalents per cytochrome c₁, while the measured content of cytochrome c₁, cytochrome b₅₆₁, cytochrome b₅₆₅, Rieske iron-sulfur protein, ubiquinone, and succinate dehydrogenase accounts for 5.0 ± 0.2 equivalents per cytochrome c₁. It is concluded that there are no unknown redox components in the cytochrome b-c₁ complex. The cytochrome b-c₁ complex (energy transduction site 2) appears to be a structural unit containing equal amounts of cytochrome c₁, cytochrome b₅₆₁, cytochrome b₅₆₆, and the Rieske iron-sulfur protein.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

Inhibition of Respiration in Prototheca zopfii by Light

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a₃. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The α-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a₃ was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.     

Effect of phospholipid on the redox potential and enzymic properties of cytochromes b.

The effects of phospholipid on the redox behavior of b cytochromes in succinate-cytochrome c reductase, the cytochrome b-c₁ complex, and an isolated cytochrome b preparation were investigated by the oxidative and reductive titrations. Three E_m values of cytochrome b were observed in the phospholipid-sufftcient and -depleted succinate-cytochrome c reductase. Their midpoint potentials at pH 7.4 are 75, 75, and ?100 mV for the sufficient and 10, ?30, and ?160 mV for the depleted reductase. The molar distribution of the b cytochromes of these E_m values correspond to 30, 30, and 40%, respectively. The E_m values of the isolated cytochrome b preparations were not affected by addition of phospholipids. The isolated b preparation contained two components of equal concentration with E_m values of ?85 and ?200 mV. No direct correlation between enzymic activity and the amount of high potential b cytochromes present in the systems was demonstrated. Very little difference was observed in redox behavior of b cytochromes between the aged inactive preparations of phospholipid-depleted reductase and that of freshly prepared reconstitutively active enzyme.     

The electron-transport chains of the obligate methylotroph Methylophilus methylotrophus

The cytochrome complement of Methylophilus methylotrophus and its respiratory properties were determined during batch culture and in continuous culture under conditions of methanol-, nitrogen- and O₂-limitation. About 35% of the cytochrome c produced by the bacteria was released into the growth medium, and of the remaining cytochrome c about half was membrane-bound and half was soluble. Two cytochromes c were always present on membranes (redox potentials 375mV and 310mV), and these probably correspond to the soluble cytochromes c described previously [Cross & Anthony (1980) Biochem. J. 192, 421–427]. A third minor component of cytochrome c (midpoint potential 356mV) was only detected on membranes of methanol-limited bacteria. M. methylotrophus always contained two membrane-bound cytochromes b with α-band absorption maxima of about 556 and 563nm (measured at room temperature) and midpoint potentials of 110 and 60mV respectively. There appeared to be relatively more of the cytochrome b₅₆₃ in methanol-limited bacteria. A third b-type cytochrome with an α-band absorption maximum at 558 (at 77K) reacted with CO and had a high midpoint redox potential (260mV); it is thus a potential oxidase and hence is called cytochrome o. The roles of these cytochromes in electron transport were confirmed by investigating the patterns of respiratory inhibition. It is proposed that two cytochromes are physiological oxidases: cytochrome a+a₃, which is present only in methanol-limited conditions, and the cytochrome o, which is induced 10-fold in conditions of methanol excess. Schemes for electron transport from methanol and NAD(P)H to O₂ in M. methylotrophus under various limitations are proposed. Spectra and potentiometric titrations of cytochromes in whole cells and membranes of M. methylotrophus grown under various nutrient limitations have been deposited as Supplementary Publication SUP 50111 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c₁ III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c₁ complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c₁ complex was separated into cytochrome b-c₁ III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K_m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c₁ III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

Evidence of ubisemiquinone radicals in electron transfer at the cytochromes b and c1 region of the cardiac respiratory chain

A highly purified reduced ubiquinone-cytochrome c reductase preparation (the b-c₁III complex) has been made. The b-c₁III complex is not reconstitutively active with succinate dehydrogenase. When the complex at about 10 mg/ml is reduced by succinate in the presence of catalytic (nanomolar) amounts of SDH and a ubiquinone protein (required in the succinate dehydrogenase region i.e, OP-S), a ubisemiquinone radical(s) has been detected using EPR measurements. The formation of the radical(s) is concurrent with the reduction of cytochrome b after the complete reduction of cytochrome c₁. All these rates are dependent on the amounts of succinate dehydrogenase and QP-S used. The maximal concentration of the radical formed is independent of the amounts of succinate dehydrogenase and QP-S added but dependent on the amount of succinate present. The formation of the radical and the reduction of b and c₁ by succinate requires the presence of phospholipids. Addition of thenoyltrifluoroacetone not only prevents the formation of the ubisemiquinone but also abolishes the prior formed radical and causes the reoxidation of b. Antimycin A also diminishes the radical intensity but causes only slight reoxidation of prior reduced cytochrome b. Treatment of the b-c₁III complex with α-chymotrypsin results in the diminishing of the radical formation. Consideration of all these results presented collectively indicates the existence of a ubiquinone binding protein in the b-c₁III complex preparation.     

Action de l'antimycine a sur l'activité succinoxydase des mitochondries du tubercule de Pomme de Terre,et comportement des cytochromes de type b

A kinetic study of the effect of antimycin A on succinate oxidase from plant mitochondria produced sigmoidal curves for the reduction of cytochromes b₅₆₀ and b₅₈₅ and for the inhibition of succinate oxidase. In the stationary state the interaction of the various components of the respiratory chain (flavins, ubiquinone, cytochromes…) occurs in a sequential mode which allows the application of simple equations utilizing rate constants and cytochrome concentrations. In these equations, it is assumed that there exists an excess of ubiquinone relative to other components of the respiratory chain as suggested by Kröger & Klingenberg (1970) and that the reoxidation of b cytochromes is fast. The inhibition by antimycin A, characterized by non-linear inhibition curves for succin-oxidase and inhibitor fixation in complex III on a component other than cytochrome c₁, is interpreted in terms of this model. This hypothesis presupposes the existence of an F factor between cytochrome b₅₆₀ and cytochrome c₁ as suggested by other authors. Utilizing these equations, theoretical curves for the inhibition of the reduction of cytochrome b₅₆₀ have been constructed and the results agree with the experimental data. The kinetic behavior of cytochrome b₅₆₆ during the induction of anaerobiosis suggests that it is not directly involved in the electron transfer chain but rather is either in thermodynamic equilibrium with cytochrome b₅₆₀ or in a shunt between cytochrome b₅₆₀ and factor F. From the experimental data, an equation is derived for the inhibition of the reduction of cytochrome b₅₆₆ by antimycin A. The actual effects of ATP and mClCCP on succinoxidase agree well with those predicted by the model.     

The electron transport systems of Fasciola hepatica mitochondria.

The electron transport systems of Fasciola hepatica mitochondria were investigated spectrophotometrically at room temperature and at −196°. The mitochondria were found to contain substrate reducible a-, b- and c-type cytochromes. All of the cytochrome components of the classical mammalian type of respiratory chain were present, although the concentration of cytochromes aa₃ was low. In addition to the mammalian type of respiratory chain, the Fasciola mitochondria contained a substrate reducible b-type cytochrome component (557 nm) which included a CO reactive o-type cytochrome. The results suggest that F. hepatica mitochondria contain a branched electron transport system including a mammalian type of chain and involving two terminal oxidases and at least two b-type cytochromes.