Structural transformation of apocytochrome c induced by alternating copolymers of maleic acid and alkene

Apocytochrome c interacts with two copolymers: poly(isobutylene-alt-maleic acid) (PIMA) and poly(1-tetradecene-alt-maleic acid) (PTMA). The interaction leads to apocytochrome c, a conformational change from random coil to alpha-helical structure. The alpha-helix content is influenced by the copolymer concentration, the length of alkyl chain of the copolymers, and pH of the medium. The electrostatic attraction between the copolymer and protein is an indispensable factor for the folding of the protein at acid pH. The hydrophobic interaction is an important factor over the entire pH range, especially when both the copolymer and protein carry negative charges at alkaline pH. The electrostatic and hydrophobic attractions between the copolymer and protein exclude water molecules, promoting the formation of hydrogen bonds within the helical structure. On the other hand, the hydrogen bonds formed between the ionized carboxyl of the copolymer and the amide of the protein partly restrain the formation of hydrogen bonds within the helical structure when the copolymer concentration is higher at pH 6.5 and 10.5.     

Region of cytochrome c interacting with yeast cytochrome b2: determination with singly modified carboxydinitrophenyl cytochromes c

The association and reduction reactions of ten different 4-carboxy-2,6-dinitrophenyl (CDNP) horse heart cytochromes c, singly modified at lysines 8, 13, 27, 39, 60, 72, 73, 86, 87, and 99, with Saccharomyces cerevisiae cytochrome b2 were studied to determine the region of cytochrome c interacting with cytochrome b2. In the presence of higher ratios of free cytochrome c to cytochrome b2, native cytochrome c, and the CDNP-lysine 39, 60, and 99 derivatives associated with cytochrome b2 with a binding stoichiometry close to 2:1, while CDNP-cytochromes c modified at lysines 8, 13, 27, 72, 73, 86, and 87 formed only 1:1 complexes. In the presence of lower ratios of free cytochrome c, modifications of lysines 8, 27, 86, and 87 had more inhibitory effects on the association of cytochrome c with cytochrome b2 than modifications of lysines 13, 39, 60, 72, 73, and 99. This tendency was similar to that on removal of free cytochrome c, except in the case of CDNP-lysine 13 and 73 derivatives. The rate of reduction of cytochrome c by cytochrome b2 was decreased by carboxydinitrophenylation of lysines 8, 13, 27, 72, 73, 86, and 87. In contrast, the rate of reduction of cytochrome c was not affected by modifications of lysines 39, 60, and 99. Since lysines 8, 13, 27, 72, 73, 86, and 87 are located on the front surface and lysines 39, 60, and 99 on the back side, and since different effects of modifying lysine residues located on the front surface may be interpreted in terms of effects on the complementary interaction of cytochrome c and cytochrome b2, these results indicate that the region of cytochrome c interacting with cytochrome b2 is located on the front surface of the cytochrome c molecule containing the exposed heme edge.     

Interaction of cytochrome c with cytochrome c oxidase: an understanding of the high- to low-affinity transition

The steady-state kinetics of high- and low-affinity electron transfer reactions between various cytochromes c and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparations were studied spectrophotometrically and polarographically. The dissociation constants for the binding of the first and second molecules of horse cytochrome c (I = 15 mM) are 5.10(-8) M and 1.10(-5) M, respectively, close to the spectrophotometric Km values and consistent with the controlled binding model for the interaction between cytochrome c and cytochrome oxidase (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 346-351) which postulates that the binding of a second molecule of cytochrome c weakens that of the first, resulting in low-affinity kinetics. While the Km of the polarographically assayed high-affinity reaction is comparable to that observed spectrophotometrically, the low-affinity Km is over an order of magnitude smaller and cannot be attributed to the binding of a second molecule of cytochrome c. Increasing the viscosity has no effect on the Vmax of the low-affinity reaction assayed polarographically, but increases the Km. Thus, the transition from high- to low-affinity kinetics is dependent on the frequency of productive collisions, as expected for a hysteresis model ascribing the transition to the trapping of the oxidase in a primed state for turnover. At ionic strengths above 150 mM, the rate of cytochrome c oxidation decreases without any correlation to the calculated net charge of the cytochrome c, indicating rate-limiting rearrangement of the two proteins in proximity to each other.     

Retinol induces permeability transition and cytochrome c release from rat liver mitochondria

Biological actions of retinoids on modulation of cellular gene expression by nuclear receptors are widely known. Recently, extra-nuclear effects of retinoids have been proposed, but remain to be better elucidated. Considering that retinoids induce apoptosis in tumor cells by an unknown mechanism, and that mitochondria play a key role in controlling apoptosis via cytochrome c (cyt c) release, we exposed rat liver mitochondria to 3-40 microM of retinol (vitamin A), and observed that retinol causes mitochondrial permeability transition (MPT) and cyt c release, in a concentration-dependent pattern. Increased superoxide anion generation and lipoperoxidation were also observed. Cyclosporin A or trolox co-administration reverted all parameters tested. In view of these findings, we conclude that retinol induces mitochondria oxidative damage, leading to MPT and cyt c release by opening of the permeability transition pore, thus suggesting a putative mechanism of apoptosis activation by retinol.     

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with Ki values of 4.5, 91, 200 and 330 microM, respectively. 2. The inhibition constant Ki of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme. 3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k1 of 33 M-1 . s-1 and a dissociation constant Kd of 3.9 mM. 4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M-1 . s-1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes. 5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome alpha 3-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

Kinetic and structural differences between cytochrome c oxidases from beef liver and heart

1. The cytochrome content of beef liver mitochondria differs from that of beef heart mitochondria by an eightfold lower cytochrome aa3 and a twofold lower cytochrome b and c + c1 content. 2. The kinetic properties of cytochrome c oxidases from beef liver and heart were measured with intact cytochrome c-depleted membranes, deoxycholate-dissolved membranes, and with the isolated enzymes at various cytochrome c concentrations with an oxygen electrode. Under all conditions a higher V was found for the liver enzyme, both for the low-affinity and for the high-affinity binding site for cytochrome c. Differences were also found for the Km of the two enzymes. 3. Isolated beef heart mitochondria contained about twice as much cardiolipin than beef liver mitochondria. The isolated enzymes contained one mole cardiolipin per mole of the heart enzyme, but 2 moles cardiolipin per mole of the liver enzyme. 4. By application of a high performance sodium dodecylsulfate gel electrophoretic system the two isolated enzymes could be separated into 13 different protein components, three of which (polypeptides VIa, VIIa and VIII) were found to differ in their apparent molecular weights. The functional meaning of cytochrome c oxidase isoenzymes in liver and heart is discussed.     

Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12 nm protein with interacting hemes

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2? resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15? resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c(7)-type domains (A, B, C and D) with homology to triheme cytochromes c(7). In cytochromes c(7) all three hemes are bis-His coordinated, whereas in c(7)-type domains the last heme is His-Met coordinated. The full-length GSU1996 has a 12nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a "nanowire" of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c(7)-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c(7)-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c(7)-type domains from one multiheme protein were compared.     

Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release

The mitochondrial permeability transition pore (PTP) and associated release of cytochrome c are thought to be important in the apoptotic process. Nitric oxide (NO( small middle dot)) has been reported to inhibit apoptosis by acting on a variety of extra-mitochondrial targets. The relationship between cytochrome c release and PTP opening, and the effects of NO( small middle dot) are not clearly established. Nitric oxide, S-nitrosothiols and peroxynitrite are reported to variously inhibit or promote PTP opening. In this study the effects of NO( small middle dot) on the PTP were characterized by exposing isolated rat liver mitochondria to physiological and pathological rates of NO( small middle dot) released from NONOate NO( small middle dot) donors. Nitric oxide reversibly inhibited PTP opening with an IC(50) of 11 nm NO( small middle dot)/s, which can be readily achieved in vivo by NO( small middle dot) synthases. The mechanism involved mitochondrial membrane depolarization and inhibition of Ca(2+) accumulation. At supraphysiological release rates (>2 micrometer/s) NO( small middle dot) accelerated PTP opening. Substantial cytochrome c release occurred with only a 20% change in mitochondrial swelling, was an early event in the PTP, and was also inhibited by NO( small middle dot). Furthermore, NO( small middle dot) exposure resulted in significantly lower cytochrome c release for the same degree of PTP opening. It is proposed that this pathway represents an additional mechanism underlying the antiapoptotic effects of NO( small middle dot).     

The effects of copper depletion on structural aspects of cytochrome c oxidase

The removal of copper from beef heart cytochrome c oxidase by either dialysis against potassium cyanide or by treatment with bathocuproine sulfonate produced changes in the enzyme which are indicative of a spin state transition. In the Soret region of the CD spectrum copper depletion of the enzyme caused a significant decrease in amplitude in combination with a red shift of the peak maximum for oxidized samples, while reduced copper-depleted samples exhibited decreased amplitude and a blue shift of the peak maximum. In the magnetic CD spectra of oxidized copper-depleted samples the peak at 420 nm was shifted to lower wave-length along with a significant increase in amplitude. In reduced samples the peak at 446 nm exhibited a slight red shift concomitant with a substantial decrease in amplitude. The conformational changes indicated by the CD and magnetic CD spectra when copper is removed from the enzyme were supported by the EPR spectra of the NO complex of the reduced copper-depleted enzyme. The removal of copper from cytochrome c oxidase caused the NO complex to exhibit a 3-line splitting pattern of gz in the EPR spectrum instead of the 9 lines seen in the NO complex of the native enzyme. When [15N]NO was used, a 2-line pattern was seen at gz when copper was removed from the enzyme. The changes in the CD and magnetic CD spectra and in the EPR spectra of the NO derivatives of cytochrome c oxidase can be explained by the rearrangement of the axial ligands to iron in cytochrome a3 as a result of copper depletion. These results emphasize the close structural interdependence of the metallic components of this enzyme.     

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane

The mechanisms of Ca2+-induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl-based media 1.4 micromol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+-induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 microM glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+ in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

Interaction of cytochrome c with cytochrome c oxidase: an NMR study on two soluble fragments derived from Paracoccus denitrificans

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase. Comparison of [(15)N,(1)H]-TROSY spectra of the [(15)N]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552). The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.     

A novel type of monoheme cytochrome c: biochemical and structural characterization at 1.23 A resolution of rhodothermus marinus cytochrome c

Monoheme cytochromes of the C-type are involved in a large number of electron transfer processes, which play an essential role in multiple pathways, such as respiratory chains, either aerobic or anaerobic, and the photosynthetic electron transport chains. This study reports the biochemical characterization and the crystallographic structure, at 1.23 A resolution, of a monoheme cytochrome c from the thermohalophilic bacterium Rhodothermus marinus. In addition to an alpha-helical core folded around the heme, common for this type of cytochrome, the X-ray structure reveals one unusual alpha-helix and a unique N-terminal extension, which wraps around the back of the molecule. Based on a thorough structural and amino acid sequence comparison, we propose R. marinus cytochrome c as the first characterized member of a new class of C-type cytochromes.     

Isolation and sequence of the structural gene for cytochrome c oxidase subunit VI from Saccharomyces cerevisiae

Using synthetic oligodeoxyribonucleic acid probes we have identified and isolated COX6, the structural gene for subunit VI of cytochrome c oxidase from Saccharomyces cerevisiae. The nucleotide sequence of COX6 predicts an amino acid sequence, for the mature subunit VI polypeptide, which is in perfect agreement with that determined previously. The nucleotide sequence of COX6 also predicts that subunit VI is derived from a precursor with a highly basic 40-amino acid NH2-terminal presequence. This precursor has been observed after in vitro translations programmed by yeast poly(A+)RNA. Northern blot analysis of poly(A+) RNA from strain D273-10B reveals that COX6 is homologous to three RNAs of 1800, 900, and 700 bases in length. By means of Southern blot analysis, the cloned gene was shown to be co-linear with yeast chromosomal DNA and to exist in a single copy in the yeast genome. An additional open reading frame, consisting of 82 codons, terminates 22 codons upstream from COX6. It is "in frame" with the COX6 coding region.     

NMR study of the conformational transition of cytochrome c upon the displacement of Met80 by exogenous ligand: structural and magnetic characterization of azidoferricytochrome c

As the exogenous ligand-cytochrome c complexes were purported to represent models for the unfolding intermediate of cytochrome c, NMR spectroscopy has been utilized to study the azide adduct of horse heart cytochrome c. The structure of azidoferricytochrome c was modeled by restrained energy minimization using paramagenetic pseudocontact shifts as constraints. The bound azide moiety was found to be tilted approximately 15 degrees from the heme normal. The displacement of Met80 by the exogenous azide molecule causes large structural rearrangement in the distal cavity. Furthermore, the conformation transition associated with the swing out of the loop containing Met80 and the shift of the 50s-helix increases the solvent accessibility of the heme group. To elucidate the heme electronic structure of the complex, the paramagnetic 13C shifts were analyzed in terms of a model based on the pi molecular orbitals of the heme under perturbed D(4) symmetry. It turned out that the His-Fe bonding provides the protein constraint that orients the in-plane anisotropy in the complex. The electronic properties are in accordance with the calculated magnetic susceptibility anisotropy and the structural information.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.