Cytochrome c interaction with membranes. Interaction of cytochrome c with isolated membrane fragments and purified enzymes

The midpoint redox potential of cytochrome c and the electron paramagnetic resonance spectra of nitroxide labeled cytochromes c were measured as a function of binding to purified cytochrome c oxidase, cytochrome c peroxidase, cytochrome b₅ and succinate—cytochrome c reductase. The midpoint redox potential of horse heart cytochrome c is lowered in the presence of cytochrome c oxidase and succinate-cytochrome c reductase, but is unchanged in the presence of cytochrome c peroxidase or cytochrome b₅. Further evidence of binding is afforded by an increase in correlation time, T_c, of the spin-labeled cytochrome c at methionine 65 upon binding to cytochrome c peroxidase, cytochrome c oxidase and succinate—cytochrome c reductase. The changes in midpoint redox potential and electron paramagnetic resonance spectrum of the spin-labeled derivative upon binding can either be the consequence of specific interaction leading to formation of ES complexes, or it can be due to nonspecific electrostatic interaction between positively charged groups on cytochrome c and negatively charged groups on the isolated cytochrome preparations.     

Cytochrome c interaction with membranes. I. Use of a fluorescent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes

Quenching of 12-(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS³ fluorescence is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids.Addition of cytochrome c to cytochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytochrome c-depleted mitochondria is 1.8 × 10⁶m, while the affinity constant for reduced cytochrome c is 0.5 × 10⁶m. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance with midpoint potential differences between the bound and free forms.     

Cytochrome c interaction with membranes. Formylated cytochrome c.

A single species of tryptophan-59 formylated cytochrome c with a half-reduction potential of 0.085 ± 0.01 V at pH 7.0 was used to study its catalytic and functional properties. The spectral properties of the modified cytochrome show that the 6th ligand position is open to reaction with azide, cyanide, and carbon monoxide. Formylated cytochrome c binds to cytochrome c depleted rat liver and pigeon heart mitochondria with the precise stoichiometry of two modified cytochrome c molecules per molecule of cytochrome a (K_D of approx 0.1 μm). Formylated cytochrome c was reducible by ascorbate and was readily oxidized by cytochrome c oxidase. The apparent K_m value of the oxidase for the formylated cytochrome c was six times higher than for the native cytochrome and the apparent V was smaller. Formylated cytochrome c does not restore the oxygen uptake in C-depleted mitochondria but inhibits, in a competitive manner, the oxygen uptake induced by the addition of native cytochrome c. Formylated cytochrome c was inactive in the reaction with mitochondrial NADH-cytochrome c reductase but was able to accept electrons through the microsomal NADPH-cytochrome c reductase.     

Cytochrome c interactions with membranes. A photoaffinity-labeled cytochrome c.

The aryl azide, 2,4-dinitro-5-fluorophenylazide, was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c, with one to two dinitroazidophenyl groups per mole of the enzyme, has a half-reduction potential the same (± 10 mV) as native cytochrome c. The dissociation constant for the modified cytochrome c from cytochrome c-depleted mitochondrial membranes and the apparent K_m for the reaction with cytochrome c oxidase were each five to six times greater than the values for native cytochrome c. Irradiation of cytochrome c-depleted mitochondrial membranes supplemented with an excess of photoaffinity-labeled cytochrome c resulted in covalent binding of the derivative to the mitochondrial membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on agarose, Bio-Gel A, showed that labeled cytochrome c was bound covalently to cytochrome c oxidase in a 1:1 molar complex. The covalently linked cytochrome c-cytochrome c oxidase complex was active in mediating the electron transfer between N,N,N′,N′-tetramethyl-p-phenylenediamine/ascorbate and the oxidase.     

The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis

While many studies have focused on cytochrome c release from mitochondria, little attention has been given to the specific interaction between cardiolipin (CL) and cytochrome c, the breaching of which likely represents a critical event in the initiation of mitochondrially mediated apoptosis. Mounting evidence suggests that a decrease in the level of CL affects cytochrome c binding to the inner membrane, thus leading to higher levels of soluble cytochrome c in the mitochondrial intermembrane space. Among the factors known to affect CL levels are thyroid status, plasma concentrations of free fatty acids, Ca²⁺ dysregulation, and reactive oxygen species (ROS). These factors, especially Ca²⁺ and ROS, have long been recognized as triggers of cell death and, more recently, as modulators of mitochondrially mediated apoptosis. In this review, we discuss the significance of the disruption of the CL-cytochrome c interaction for cytochrome c release and apoptosis.     

Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence

In addition to its electron transfer activity, cytochrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modification of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial membrane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV–visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations permitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine protonation is relatively narrow and is consistent with the mitochondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main candidate to replace methionine in the non-native cytochrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment.     

Nitric Oxide Binds to the Proximal Heme Coordination Site of the Ferrocytochrome c/Cardiolipin Complex: FORMATION MECHANISM AND DYNAMICS*

Mammalian mitochondrial cytochrome c interacts with cardiolipin to form a complex (cyt. c/CL) important in apoptosis. Here we show that this interaction leads to structural changes in ferrocytochrome c that leads to an open coordinate site on the central iron, resulting from the dissociation of the intrinsic methionine residue, where NO can rapidly bind (k = 1.2 × 10⁷ m⁻¹ s⁻¹). Accompanying NO binding, the proximal histidine dissociates leaving the heme pentacoordinate, in contrast to the hexacoordinate nitrosyl adducts of native ferrocytochrome c or of the protein in which the coordinating methionine is removed by chemical modification or mutation. We present the results of stopped-flow and photolysis experiments that show that following initial NO binding to the heme, there ensues an unusually complex set of kinetic steps. The spectral changes associated with these kinetic transitions, together with their dependence on NO concentration, have been determined and lead us to conclude that NO binding to cyt. c/CL takes place via an overall scheme comparable to that described for cytochrome c′ and guanylate cyclase, the final product being one in which NO resides on the proximal side of the heme. In addition, novel features not observed before in other heme proteins forming pentacoordinate nitrosyl species, include a high yield of NO escape after dissociation, rapid (<1 ms) dissociation of proximal histidine upon NO binding and its very fast binding (60 ps) after NO dissociation, and the formation of a hexacoordinate intermediate. These features all point at a remarkable mobility of the proximal heme environment induced by cardiolipin.     

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H₂O₂, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H₂O₂-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H₂O₂-induced liposome leakage was abolished by cyanide presumably competing with H₂O₂ for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H₂O₂ permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H₂O₂, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.     

Studies on the orientations of the mitochondrial redox carriers II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated,oriented mitochondrial multilayers

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c₁, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the “tightly bound” mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90°.     

Purification, physicochemical properties, and localization of cytochrome c in energy-transducing membranes from Mycobacterium phlei.

Cytochrome c from Mycobacterium phlei has been isolated and purified to homogeneity using an isoelectric focusing technique. The purified cytochrome c has a molecular weight of 12,600 ± 400 and exhibits an isoelectric point (pI) of 4.7 ± 0.05. The amino acid composition of cytochrome c shows a higher proportion of valine and arginine residues and a greatly reduced content of lysine residues when compared to Bacillus subtilis cytochrome c. This imparts less acidic character to the cytochrome c from M. phlei. The cytochrome c from M. phlei acts as the most effective electron acceptor for M. phlei NADH-cytochrome c reductase, while yeast and horse heart cytochrome c are not as efficient electron acceptors. The absence of correlation between the oxidation-reduction potential with the observed activity of NADH-cytochrome c reductase activity indicates that the electrochemical potential is not a sufficient determinant for bacterial cytochrome c function. In order to obtain information concerning the topology of respiratory components, two membrane systems from M. phlei were used; ghost preparations in which the membrane is oriented rightside out as in whole cells and membrane vesicles in which membranes are oriented inside out. Labeling of protoplast ghosts and membrane vesicles with lactoperoxidase-catalyzed iodination reveals that cytochrome c is localized on the outer membrane of protoplast ghosts, which is similar to that observed in mammalian mitochondria. The results also show that cytochrome c from M. phlei binds preferentially to basic phospholipids and not to neutral or acidic phospholipids. Scatchard analysis of the binding of cytochrome c to phosphatidyl ethanolamine shows high affinity (K_a of 3.79 × 10⁵M^?1) and low affinity (K_a of 3.75 × 10⁴M^?1) binding.     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

Cytochrome c interaction with membranes. The enthalpy of oxidation of cytochromes c, c2, and a1,2.

The enthalpy of oxidation of horse-heart cytochrome c bound to phospholipid vesicles was found to be 14.6 ± 0.3 kcal/mole at 25 °C, pH 7.0, equal to the value for oxidation of the free form of the cytochrome. The affinity constants for binding of the reduced and oxidized forms of cytochrome c were the same at 4 °C and 30 °C, indicating that ΔH ° of binding contributes negligibly to the overall enthalpy of oxidation of the bound cytochrome c. The free energy (ΔG °′) of oxidation of the bound cytochrome c was 1.3 kcal/mole smaller than that for the free form, the difference being due to the change in entropy favoring the oxidized state of the cytochrome in the bound state. Measurement of the ΔH °′ for the oxidation of cytochrome a relative to the ferri/ferrocyanide couple shows it to be the same, within the limits of experimental error to that for the oxidation of cytochrome c.     

Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN*

The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (<r.m.s. deviation>_bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (<r.m.s. deviation>_bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.     

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.     

Three-dimensional structures of cytochrome c oxidase vesicle crystals in negative stain

We have investigated the structure of cytochrome c oxidase vesicle crystals by analysis at 20 Å resolution of electron micrographs of negatively stained specimens. The map clearly shows the shape of the part of the cytochrome c oxidase molecule which protrudes from the lipid bilayer. On the side of the membrane corresponding to the cytoplasmic face of the mitochondrial inner membrane, the molecule projects over 50 Å into solution. About half of the mass of the protein is in this domain, which contains the cytochrome c binding site. On the side of the membrane corresponding to the matrix face, no features are observed, which at this resolution means the protein protrudes less than 20 Å. In vesicle crystals, and probably in the mitochondrion, cytochrome c oxidase monomers are closely paired as dimers, with a clear cleft showing the boundary between monomers.     

Properties of rat liver mitochondria with intermembrane Cytochrome c.

When rat liver mitochondria were suspended in 0.15 m KCl, the cytochrome c appeared to be solubilized from the binding site on the outside of the inner membrane and trapped in the intermembrane space. When the outer membrane of these mitochondria was disrupted with digitonin at a digitonin concentration of 0.15 mg/mg of protein, the solubilized cytochrome c could be released from mitochondria along with adenylate kinase. When mitochondria were suspended in 0.15 m KCl instead of 0.33 m sucrose, the $\text{ADP}\text{O}$ ratio observed with succinate, β-hydroxybutyrate, malate + pyruvate or glutamate as substrates was little affected. A number of cycles of State 4-State 3-State 4 with ADP was observed. The respiratory control ratios, however, were decreased, particularly when glutamate was used as the substrate. Cytochrome c oxidase activity was also decreased to 55% when assayed using ascorbate + N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD) as substrates. Suspension of mitochondria in 0.15 m KCl resulted in an enhancement of the very low NADH oxidation by intact mitochondria and a twofold enhancement of sulfite oxidation. Trapped cytochrome c in outer membrane vesicles prepared from untreated and trypsin-treated intact mitochondria was found to be readily reduced by NADH and suggests that some cytochrome b₅ is located on the inner surface of the outer membrane. The enhanced NADH oxidase could therefore reflect the ability of cytochrome c to mediate intermembrane electron transport. The enhanced sulfite oxidase activity was sensitive to cyanide inhibition and coupled to oxidative phosphorylation ($\text{ADP}\text{O}\text{< 1}$) unlike the activity of mitochondria in sucrose medium. These results suggest that free cytochrome c in the intermembrane space can mediate electron transfer between the sulfite oxidase and the inner membrane.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Heme-heme interaction in cytochrome c oxidase: the cooperativity of the hemes of cytochrome c oxidase as evidenced in the reaction with CO

Isolated and purified cytochrome c oxidase from beef heart muscle mitochondria (Kuboyama et al. (1972) J. Biol. Chem.247, 6375–6383) is shown to be very similar to the hemoprotein in situ with respect to its EPR absorption properties and the half-reduction potentials of the hemes and copper. The half-reduction potentials of cytochromes a and a₃ in the purified cytochrome c oxidase are 205 mV and 360 mV, respectively, and these values are the same in the presence and absence of cytochrome c.Low-temperature EPR spectra show that the binding of CO to reduced cytochrome a₃ changes the oxidized cytochrome a from high spin (g 6) to low spin (g 3). In samples at 5–8 °K the photodissociation of the reduced cytochrome a₃CO compound shifts the spectrum of the oxidized low-spin cytochrome a to a lower g value and converts approximately 5% of the low-spin form to a high-spin form. The heme-heme interaction demonstrated in this reaction is very fast as evidenced by the fact that even at 5 °K the measured change in oxidized cytochrome is complete within 5 msec.     

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.