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.     

Structural transformation of cytochrome c and apo cytochrome c induced by sulfonated polystyrene

The structural transformation of cytochrome c (cyt c) and its heme-free precursor, apo cyt c, induced by negatively charged sulfonated polystyrene (SPS) with different charge density (degree of sulfonation) and chain length was studied to understand the factors that influence the folding and unfolding of the protein. SPS forms stable transparent nanoparticles in aqueous solution. The hydrophobic association of the backbone chain and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The binding of cyt c to negatively charged SPS particles causes an extensive disruption of the native compact structure of cyt c: the cleavage of Fe-Met80 ligand, about 40% loss of the helical structure, and the disruption of the asymmetry environment of Trp59. On the other hand, SPS particle-bound apo cyt c undergoes a conformational change from the random coil to alpha-helical structure. The folding of apo cyt c in SPS particles was influenced by pH and ionic strength of the solution, SPS concentration, and the degree of sulfonation and chain length of SPS. The folding can reach more than 90% of the alpha-helix content of native cyt c in solution. Poly(sodium 4-styrenesulfonate) (PSS), which is 100% sulfonated polystyrene and cannot form hydrophobic cores in the solution, induces only two-thirds of the alpha-helix content compared with SPS. It appears that the electrostatic interaction between PSS/SPS and apo cyt c induces an early partially folded state of apo cyt c. The hydrophobic interaction between nonpolar residues in apo cyt c and the hydrophobic cores in SPS particles extends the alpha-helical structure of apo cyt c.     

Modelling of the human papillomavirus type 16 E5 protein

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7-9.9, at which 68-74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.     

Interaction between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: effects of charge-modifying mutations on binding and electron transfer.

The electrostatic interactions governing binding and electron transfer from cytochrome c(2) (cyt c(2)) to the reaction center (RC) from the photosynthetic bacteria Rhodobacter sphaeroides were studied by using site-directed mutagenesis to change the charges of residues on the RC surface. Charge-reversing mutations (acid --> Lys) decreased the binding affinity for cyt c(2). Dissociation constants, K(D) (0.3--250 microM), were largest for mutations of Asp M184 and nearby acid residues, identifying the main region for electrostatic interaction with cyt c(2). The second-order rate constants, k(2) (1--17 x 10(8) M(-1) s(-1)), increased with increasing binding affinity (log k(2) vs log 1/K(D) had a slope of approximately 0.4), indicating a transition state structurally related to the final complex. In contrast, first-order electron transfer rates, k(e), for the bound cyt did not change significantly (<3-fold), indicating that electron tunneling pathways were unchanged by mutation. Charge-neutralizing mutations (acid --> amide) showed changes in binding free energies of approximately 1/2 the free energy changes due to the corresponding charge-reversing mutations, suggesting that the charges in the docked complex remain well solvated. Charge-enhancing mutations (amide --> acid) produced free energy changes of the same magnitude (but opposite sign) as changes due to the charge-neutralizing mutations in the same region, indicating a diffuse electrostatic potential due to cyt c(2). A two-domain model is proposed, consisting of an electrostatic docking domain with charged surfaces separated by a water layer and a hydrophobic tunneling domain with atomic contacts that provide an efficient pathway for electron transfer.     

Conformation and reactivity of DNA in the complex with protein. IV. Circular dichroism of poly-L-histidine model complexes with DNA polymers and specificity of the interaction.

The CD study of the DNA-poly-L-histidine complex at high degree of protonation revealed that complex formation is already observable at 2 M NaCl. The influence of salt together with 5 M urea suggests that in addition to electrostatic interactions probably hydrogen bonding may favour specific complexes. Affinity of protonated histidines to AT-rich regions is strongly supported by the complexes formed with (dA.dT)-containing polymers. The psi-type structure occurs with poly(dA-dT)-poly(dA-dT) while poly(dA)-poly(dT) is restricted to form a similar psi-state on interaction with highly protonated poly-L-histidine. Differences in the helix winding properties due to variation in the sequence is suggested as a possible factor in the formation of the psi-type complexes. The mechanism of interaction including hydrogen bonding of histidine side-chains with an AT pair at high degree of protonation and with GC-regions at lower degree of protonation in the polypeptide structure is discussed.     

Brownian dynamics simulations of the interaction of Chlamydomonas cytochrome f with plastocyanin and cytochrome c6

The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments.     

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.     

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.     

Cytochrome c forms complexes and is partly reduced at interaction with GPI-anchored alkaline phosphatase

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7–9.9, at which 68–74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.     

Identification of precise electrostatic recognition sites between cytochrome c6 and the photosystem I subunit PsaF using mass spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.     

Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models

Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon.     

Electrostatic properties of cytochrome f: implications for docking with plastocyanin.

The electrostatic properties of cytochrome f (cyt f), a member of the cytochrome b6f complex and reaction partner with plastocyanin (PC) in photosynthetic electron transport, are qualitatively studied with the goal of determining the mechanism of electron transfer between cyt f and PC. A crystal structure for cyt f was analyzed with the software package GRASP, revealing a large region of positive potential generated by a patch of positively charged residues (including K58, K65, K66, K122, K185, K187, and R209) and reinforced by the iron center of the heme. This positive field attracts the negative charges of the two acidic patches on the mobile electron carrier PC. Three docked complexes are obtained for the two proteins, based on electrostatic or hydrophobic interactions or both and on steric fits by manual docking methods. The first of these three complexes shows strong electrostatic interactions between K187 on cyt f and D44 on PC and between E59 on PC and K58 on cyt f. Two other manually docked complexes are proposed, implicating H87 on PC as the electron-accepting site from the iron center of cyt f through Y1. The second complex maintains the D44/K187 cross-link (but not the E59/K58 link) while increasing hydrophobic interactions between PC and cyt f. Hydrophobic interactions are increased still further in the third complex, whereas the link between K187 on cyt f and D44 on PC is broken. The proposed reaction mechanism, therefore, involves an initial electrostatic docking complex that gives rise to a nonpolar attraction between the regions surrounding H87 on PC and Y1 on cyt f, providing for an electron-transfer active complex.     

Surface-enhanced resonance Raman spectroscopy and spectroscopy study of redox-induced conformational equilibrium of cytochrome c adsorbed on DNA-modified metal electrode

The redox-induced conformational equilibrium of cytochrome c (cyt c) adsorbed on DNA-modified metal electrode and the interaction mechanism of DNA with cyt c have been studied by electrochemical, spectroscopic and spectroelectrochemical techniques. The results indicate that the external electric field induces potential-dependent coordination equilibrium of the adsorbed cyt c between its oxidized state (with native six-coordinate low-spin and non-native five-coordinate high-spin heme configuration) and its reduced state (with native six-coordinate low-spin heme configuration) on DNA-modified metal electrode. The strong interactions between DNA and cyt c induce the self-aggregation of cyt c adsorbed on DNA. The orientational distribution of cyt c adsorbed on DNA-modified metal electrode is potential-dependent, which results in the deviation from an ideal Nernstian behavior of the adsorbed cyt c at high electrode potentials. The electric-field-induced increase in the activation barrier of proton-transfer steps attributed to the rearrangement of the hydrogen bond network and the self-aggregation of cyt c upon adsorption on DNA-modified electrode strongly decrease the interfacial electron transfer rate. In addition, the strongly Coulombic interactions between DNA and cyt c only disturb the microenvironment of the heme, and do not affect the states of heme ligation and spin. The secondary structure of the adsorbed cyt c is retained, while the conformation of DNA is changed from the B form DNA to A form DNA.     

Stable submolecular folding units in a non-compact form of cytochrome c.

Studies of structure, dynamics, and stability of cytochrome c (cyt c) at low pH in a non-compact pre-molten globule state indicate that the protein contains submolecular folding units that are independently stable. In high salt, acid cyt c (pD 2.2; where D is deuterium) is nearly as compact as the native form. Nuclear magnetic resonance (n.m.r.) line broadening typical of the molten globule form is seen, indicating loosened packing and increased mobility not only for side-chains but also for the main chain. As NaCl concentration is decreased below 0.05 M, cyt c expands due to the deshielding of electrostatic repulsions, attaining a linear extent perhaps double that of the native protein (viscosity, fluorescence). In the extended form, tertiary structural hydrogen bonds are largely broken (hydrogen exchange rate), some normally buried parts of the protein are exposed to water (fluorescence), and many of the native side-chain contacts must be lost. Nevertheless, almost all of the helical content is retained (circular dichroism). The helices involve the same amino acid residues that are helical in the native state (hydrogen exchange labeling monitored by 2-dimensional n.m.r.). The equilibrium constant for helix formation at 20 degrees C (0.02 M-NaCl, pD 2.2) is about 10 (hydrogen exchange rate), even though the individual helical segments when isolated have little or no structure. Additional experiments were done to check assumptions and calibrate parameters that underlie the hydrogen exchange analysis of protein folding. These results indicate that the native-like helical segments in the expanded non-globular form of cyt c exist as part of somewhat larger submolecular folding units that possess significant equilibrium stability. Results from equilibrium and kinetic studies of protein folding support the generality of this conclusion. This view is contrary to the two-state paradigm for equilibrium folding and inconsistent with the idea that side-chain packing constraints determine folding motifs. The result suggests an extension of the thermodynamic hypothesis for protein structure to kinetic folding processes, so that the amino acid code for equilibrium and kinetic folding may be the same, and also seems pertinent to the biological evolution of contemporary protein structures.     

Cytochrome c redox state influences the binding and release of cytochrome c in model membranes and in brain mitochondria

Cytochrome c (cyt c), a component of the respiratory chain, promotes apoptosis when released into the cytosol. Cyt c anchorage within mitochondria depends on cardiolipin (CL). Detachment and release have been related to CL loss and peroxidation. We report that NaN₃-dependent complex IV inhibition, accompanied by impairment of respiration, resulted in cyt c release. Contrarily, inhibition of respiration upstream cyt c with complex I and III inhibitors was not accompanied by the release of the protein, despite CL decrease and monolyso-CL increase. No CL changes and H₂O₂ formation were observed by inhibiting complex IV. In cyt c–CL liposomes, breaching cyt c–CL hydrophilic interactions produced a higher release of the reduced, compared to the oxidized form, suggesting that the hydrophobic component of cyt c–CL binding is prevalent in the oxidized form. Free or liposome-reconstituted cyt c was able to form fatty acid–protein complexes (palmitate < linoleate < oleate) only in its reduced form. We hypothesize that reduced cyt c–fatty acid binding favors the dislocation of the protein from anchoring CL. A mechanism for cyt c release independent of CL peroxidation by H₂O₂ is feasible. It could weaken the hydrophobic component of cyt c–CL interactions and might function following complex IV inhibition or in oxygen lack, both conditions producing accumulation of reduced cyt c and free fatty acids.     

ATP specifically drives refolding of non-native conformations of cytochrome c

An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.     

Cytochrome c superstructure biocomposite nucleated by gold nanoparticle: thermal stability and voltammetric behavior

The thermal stability of cytochrome c (cyt c) after Au-nanoparticle-directed association has been studied by various spectroscopic (electronic absorption, resonance Raman, and circular dichroism) and electrochemical methods. The results show that the thermal stability of the Au-cyt c superstructure biocomposite formed by the electrostatic and hydrophobic interactions among the associated proteins increases significantly. It is mainly caused by strong hydrophobicity of the associated cyt c in Au-cyt c superstructure at high temperature, which results from the compact secondary structure and the packing of hydrophobic side chains around the Trp 59 and heme. In addition, the formation of bis-His configuration of heme is facilitated by the tightly self-associated state of cyt c in the Au-cyt c superstructure. The electrostatic coupling of the opposite charges among shells of the adsorbed proteins due to the formation of the superstructure biocomposite can reduce repulsions among the same charges in protein. These factors are also important for enhancing the stability of the associated cyt c. Furthermore, the voltammetric behavior of Au-cyt c at DNA modified glassy carbon electrode has been investigated for extending the application of Au-cyt c.     

Substitution of the sixth axial ligand of Rhodobacter capsulatus cytochrome c1 heme yields novel cytochrome c1 variants with unusual properties.

The cytochrome (cyt) c1 heme of the ubihydroquinone:cytochrome c oxidoreductase (bc1 complex) is covalently attached to two cysteine residues of the cyt c1 polypeptide chain via two thioether bonds, and the fifth and sixth axial ligands of its iron atom are histidine (H) and methionine (M), respectively. The latter residue is M183 in Rhodobacter capsulatus cyt c1, and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c1 [Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b6f complex, the sixth axial ligand is provided by the amino group of the amino terminal tyrosine residue. To further pursue our investigation on the role played by the sixth axial ligand in heme-protein interactions, novel cyt c1 variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c1 mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc1 complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c1 polypeptides, but yielded nonfunctional bc1 complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambdamax) and decreased redox midpoint potential values (Em7). The availability and future detailed studies of these stable cyt c1 mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.     

Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity

Upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL may act as a switch to regulate cyt c's mitochondrial functions. Using cyclic voltammetry and equilibrium redox titrations, we show that the redox potential of cyt c shifts negatively by 350-400 mV upon binding to CL-containing membranes. Consequently, functions of cyt c as an electron transporter and cyt c reduction by Complex III are strongly inhibited. Further, CL/cyt c complexes are not effective in scavenging superoxide anions and are not effectively reduced by ascorbate. Thus, both redox properties and functions of cyt c change upon interaction with CL in the mitochondrial membrane, diminishing cyt c's electron donor/acceptor role and stimulating its peroxidase activity.