Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain

The binding of cytochrome c to the cytochrome bc₁ complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc₁ complex were measured. Mixtures of bc₁ complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dualwavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 13 derivative produced a diminution of the polypeptide of the bc₁ complex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c₁, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc₁ complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.     

The interaction between heme and protein in cytochrome c1

The optical spectrum of reduced bovine cytochrome c₁ at 77 K shows a fine splitting of the β-band, which is indicative of the native conformation of the protein. At room temperature, this conformation is reflected in an absorbance band at 530 nm. The exposure of the heme of ferrocytochrome c₁, investigated by means of solvent-perturbation spectroscopy, appears to be extremely sensitive to temperature and SH reagents bound to the oxidized protein. Addition of combinations of potential ligands to the isolated tryptic heme peptide of cytochrome c₁ reveals that only a mixture of methionine and cysteine (or their equivalents) generates a β-band at 77 K which is identical in shape to that of native cytochrome c₁. In the EPR spectrum of a complex of ferrocytochrome c₁ and nitric oxide at pH 10.5, no hyperfine splitting derived from a second ligated nitrogen atom could be detected. The results indicate that methionine and cysteine are the axial ligands of heme in cytochrome c₁. The EPR spectrum of isolated ferricytochrome c₁ is that of a low-spin heme iron compound with a g_z value of 3.36 and a g_y value of 2.04.     

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.     

Isolation of QP-C and reconstitution of the QH2-c reductase

A ubiquinone protein, QP-C, which acts in the cytochrome $\text{b}\text{-}\text{c}$₁ region has been solubilized. The isolated QP-C shows one band of molecular weight 15,000 in polyacrylamide gel electrophoresis in sodium dodecyl sulfate and isoelectric focusing at the isoelectric point of pH 3.6. QP-C reconstitutes with the QP-C deficient $\text{b}\text{-}\text{c}$₁ complex to restore the OH₂-cytochrome $\text{c}$ activity and recover the EPR signal of the complex.     

Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.     

Low-temperature time-resolved spectroscopic study of the major light-harvesting complex of Amphidinium carterae

The major light-harvesting complex of Amphidinium (A.) carterae, chlorophyll-a–chlorophyll-c ₂–peridinin–protein complex (acpPC), was studied using ultrafast pump-probe spectroscopy at low temperature (60 K). An efficient peridinin–chlorophyll-a energy transfer was observed. The stimulated emission signal monitored in the near-infrared spectral region was stronger when redder part of peridinin pool was excited, indicating that these peridinins have the S₁/ICT (intramolecular charge-transfer) state with significant charge-transfer character. This may lead to enhanced energy transfer efficiency from “red” peridinins to chlorophyll-a. Contrary to the water-soluble antenna of A. carterae, peridinin–chlorophyll-a protein, the energy transfer rates in acpPC were slower under low-temperature conditions. This fact underscores the influence of the protein environment on the excited-state dynamics of pigments and/or the specificity of organization of the two pigment–protein complexes.     

Synthesis and characterization of transition metal dithiocarbamate derivatives of 1-aminoadamantane: Crystal structure of (N-adamantyldithiocarbamato)nickel(II)

Four bis(N-adamantyldithiocarbamato)metal(II) complexes [M(S₂CNHC₁₀H₁₅)₂] (M = Ni, Zn, Cd, and Hg) were prepared by metathesis of the corresponding potassium salt, K[S₂CNHC₁₀H₁₅]. Characterization of the prepared complexes shown that the presence of only one band in the region 1000 cm⁻¹ in IR spectra can be attributed to a completely symmetrical bonding of the dithiocarbamate ligand, acting in a symmetrical bidentate mode. Also the short thioureide C-N distance in X-ray analysis of Ni complex confirm the delocalization of the π electrons over the S₂CN moiety and strong double bond character.     

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.     

Components of the major urinary protein complex of inbred mice: Determination of NH2-terminal sequences and comparison with homologous components from wild mice

The major urinary protein (MUP) complex of normal inbred laboratory mice (Mus musculus musculus) is a family of three electrophoretically distinguishable components, designated 1, 2, and 3 in order of increasing anodal mobility at pH 5.5. Components 1 and 2 are under the control of a single genetic locus; the MUP complex of a given inbred strain consists of component 1 or 2 plus component 3. In this study, the urinary protein of two subspecies of Asian wild mice, Mus musculus molossinus (originally trapped in Japan) and Mus musculus castaneus (originally trapped in Thailand), was examined electrophoretically and ultracentrifugally. The MUP complex of male M. m. molossinus and M. m. castaneus sedimented at approximately the same rate as that of M. m. musculus (s ₂₀ =2.0?2.2S). It consisted of a “fast” (i.e., more anodal than component 3) and an “intermediate” component plus one or more “origin” (i.e., less anodal than component 1) components. The “fast” and “origin” components were isolated chromatographically, and NH₂-terminal sequences spanning the first 36 residues were determined. Comparison with the NH₂-terminal sequences determined for components 1, 2, and 3 isolated from the urine of BALB/c or C57BL/6 mice revealed, except for a single replacement at position 6 in the “origin” component of M. m. molossinus, no differences among the 1, 2, “origin”, and “fast” components. Component 3 was highly homologous but differed from component 1 at nine positions; its residue at position 6 was the same as that of the M. m. molossinus “origin” component.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

Sex and the fixation of single favourable mutations

Sexual populations will accumulate favourable mutations more rapidly than asexual populations. This is true if it is often the case that two different favourable mutations can be found to be spreading simultaneously through populations. It is argued here that sexual species will incorporate single favourable mutations more quickly than asexual “species”, if the latter are multi-clonal. Thus one mutation can spread to fixation within a sexual species but in an asexual “species” with N_c clones at least N_c mutations must occur if the mutation is to be subsequently found in every member of the “species”. Asexual “species” may minimise this disadvantage by evolving polyploidy or occasional episodes of hybridisation. Both are in fact common in asexual “species”.     

Effects of alcohol/water mixtures on the structure and reactivity of cytochrome c

The oxidation reaction of ferrocytochrome c (produced in situ by pulse radiolysis) by Fe(CN)^3?₆, was used to probe the effect of alcohol/water mixtures on the reactivity of the protein. Reduced cytochrome c is oxidized in a biphasic process. The relative contribution of each phase depended on: pH, alcohol concentration and temperature. pK_a values were derived from the kinetic data. These pK_a values were identical with the spectroscopic pK_a values determined under similar conditions by monitoring the 695 nm absorption band of the oxidized protein. The two phases of oxidation were therefore related to the oxidation of a relaxed and a nonrelaxed conformer of reduced cytochrome c produced in situ. A shift in the pK_a of ferricytochrome c and a retardation of the redox reactions of both the reduced and the oxidized protein were observed at low alcohol concentrations (up to 5 mol %). These low alcohol concentrations are known to affect the structure of water (Yaacobi, M. and Ben-Naim, A. (1973) J. Sol. Chem. 5, 425?443; Ben-Naim, A. (1967) J. Phys. Chem. 71, 4002?4007 and Ben-Naim, A. and Baer, S. (1964) Trans. Faraday Soc. 60, 1736?1741) but have only minor effects on the protein. Accordingly, the kinetic results are interpreted on the basis of involvement of water molecules in the reaction complex of cytochrome c with its redox substrates.     

A new method of determination of the carboxyl-terminal residue of peptides

A colorimetric method has been developed for monitoring the activation of polysaccharides with cyanogen bromide. The method is based on the “K?nig” reaction of cyanate esters with pyridine and barbituric acid to yield a redpurple colored complex with λ_max, 575 nm and an ε_M value of 15,000 M^?1cm^?1. Besides providing a means for a quantitative measurement of activation of the gel with CNBr, the method also enables us to determine (a) the extent of washing the activated gel required for the removal of the unreacted reagent; and (b) the amount and the decay of the reactive groups remaining after the coupling of the ligand to the activated gel.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Reinterpretation of the vibrational spectroscopy of the medicinal bioinorganic synthon c,c,t-[Pt(NH3)2Cl2(OH)2]

The Pt(IV) complex c,c,t-[Pt(NH₃)₂Cl₂(OH)₂] is an important intermediate in the synthesis of Pt(IV) anticancer prodrugs and has been investigated as an anticancer agent in its own right. An analysis of the vibrational spectroscopy of this molecule was previously reported (Faggiani et al., Can. J. Chem. 60:529, 1982), in which crystallographic determination of the structure of the complex permitted a site group approach. The space group, however, was incorrectly assigned. In the present study we have redetermined at high resolution crystal structures of c,c,t-[Pt(NH₃)₂Cl₂(OH)₂] and c,c,t-[Pt(NH₃)₂Cl₂(OH)₂]·H₂O₂, which makes possible discussion of the effect of hydrogen bonding on the N–H and O–H vibrational bands. The correct crystallographic site symmetry of the platinum complex in the c,c,t-[Pt(NH₃)₂Cl₂(OH)₂] structure is used to conduct a new vibrational analysis using both group-theoretical and modern density functional theory methods. This analysis reveals the nature and symmetry of the “missing band” described in the original publication and suggests a possible explanation for its disappearance.     

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.     

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).     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.