The interaction of cytochrome c (cyt c) with mitochondrial mimetic vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and heart cardiolipin (PCPECL) was investigated over the 7.4 -6.2 pH range by means of turbidimetry and photon correlation spectroscopy. In the presence of cyt c, the decrease of pH induced an increase in vesicle turbidity and mean diameter resulting from vesicle fusion as determined by a rapid decrease in the excimer/monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidylcholine (PyPC). N-acetylated cyt c and protamine, a positively charged protein, increased vesicle turbidity in a pH-independent manner, whereas albumin did not affect PCPECL vesicle turbidity. pH-dependent turbidity kinetics revealed a role for cyt c-ionizable groups with a pK a(app) of ϳ7.0. The carbethoxylation of these groups by diethylpyrocarbonate prevented cyt c-induced vesicle fusion, although cyt c association to vesicles remained unaffected. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that Lys-22, Lys-27, His-33, and Lys-87 cyt c residues were the main targets for carbethoxylation performed at low pH values (<7.5). In fact, these amino acid residues belong to clusters of positively charged amino acids that lower the pK a . Thus, at low pH, protonation of these invariant and highly conserved amino acid residues produced a second positively charged region opposite to the Lys-72 and Lys-73 region in the cyt c structure. These two opposing sites allowed two vesicles to be brought together by the same cyt c molecule for fusion. Therefore, a novel pH-dependent site associating cyt c to mitochondrial mimetic membranes was established in this study.

Recently cytochrome c has been mentioned as an important mediator in the events of cellular oxidative stress and apoptosis. To investigate the influence of charged interfaces on the conformation of cytochrome c, the CD and magnetic circular dichroic behavior of ferric and ferrous cytochrome c in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and dicetylphosphate liposomes was studied in the 300 -600 and 200 -320 nm wavelength region. EPR spectra demonstrate that the association of cytochrome c with membranes promotes alterations of the crystal field symmetry and spin state of the heme Fe 3؉ . The studies also include the effect of P i , NaCl, and CaCl 2 . Magnetic circular dichroism and CD results show that the interaction of both ferrous and ferric cytochrome c with charged interfaces promotes conformational changes in the ␣-helix content, tertiary structure, and heme iron spin state. Moreover, the association of cytochrome c with different liposomes is sensitive to the heme iron valence state. The more effective association with membranes occurs with ferrous cytochrome c. Dicetylphosphate liposomes, as a negatively charged membrane model, promoted a more pronounced conformational modification in the cytochrome c structure. A decrease in the lipid/protein association is detected in the presence of increasing amounts of CaCl 2 , NaCl, and P i , in response to the increase of the ionic strength.

It has been shown that lysosomal cysteine proteinases, specially cathepsin B, has been implicated in a variety of diseases involving tissue remodeling states, such as inflammation, parasite infection, and tumor metastasis, by degradation of extracellular matrix components. Recently, we have shown that heparin and heparan sulfate bind to papain specifically; this interaction induces an increase of its ␣-helix content and stabilizes the enzyme structure even at alkaline pH (Almeida, P. C., Nantes, I. L., Rizzi, C. C. A., Jú dice, W. A. S., Chagas, J. R., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (1999) J. Biol. Chem. 274, 30433-30438). In the present work, a combination of circular dichroism analysis, affinity chromatography, cathepsin B mutants, and fluorogenic substrate assays were used to characterize the interaction of human cathepsin B with glycosaminoglycans. The nature of the cathepsin B-glycosaminoglycans interaction was sensitive to the charge and type of polysaccharide. Like papain, heparin and heparan sulfate bind cathepsin B specifically, and this interaction reduces the loss of cathepsin B ␣-helix content at alkaline pH. Our data show that the coupling of cathepsin B with heparin or heparan sulfate can potentiate the endopeptidase activity of the cathepsin B, increasing 5-fold the half-life (t1 ⁄2 ) of the enzyme at alkaline pH. Most of these effects are related to the interaction of heparin and heparan sulfate with His 111 residue of the cathepsin B occluding loop. These results strongly suggest that heparan sulfate may be an important binding site for cathepsin B at cell surface, reporting a novel physiological role for heparan sulfate proteoglycans.Heparan sulfate is an ubiquitous glycosaminoglycan of animal cells (1). These classes of compounds are heteropolysaccharides made up of repeating units of disaccharides, an uronic acid residue, either D-glucuronic acid or L-iduronic acid, and D-glucosamine with N-and 6-O-sulfates and N-acetyl substitutions (2). Heparan sulfate occurs at the cell surface and in extracellular matrix as proteoglycans. Most of cellular heparan sulfate derives from the syndecans and glypicans proteoglycans. The syndecan family are associated with the cell membranes via transmembrane core proteins (3, 4), and the glypican family is anchored by glycosilyl phosphatidylinositolanchor core proteins (5). Also, heparan sulfate proteoglycans are present in basement membranes performing the perlecan family (6).Heparan sulfate and heparin are particular among glycosaminoglycans in their ability to bind a large number of different proteins. Heparin-like glycosaminoglycans play a complex role in the extracellular matrix, regulating a wide variety of biological process, including hemostasis, inflammation, angiogenesis, growth factors, cell adhesion, and others (7). Proteolytic enzymes control many of these biological process. Several reports in the literature have demonstrated that heparin-like glycosaminoglycans can modulate the activity of some serine proteinases and their natu...

This work compares the effect of photogenerated singlet oxygen (O 2 ( 1 ⌬ g )) (type II mechanism) and free radicals (type I mechanism) on cytochrome c structure and reactivity. Both reactive species were obtained by photoexcitation of methylene blue (MB ؉ ) in the monomer and dimer forms, respectively. The monomer form is predominant at low dye concentrations (up to 8 M) or in the presence of an excess of SDS micelles, while dimers are predominant at 0.7 mM SDS. Over a pH range in which cytochrome c is in the native form, O 2 ( 1 ⌬ g ) and free radicals induced a Soret band blue shift (from 409 to 405 nm), predominantly. EPR measurements revealed that the blue shift of the Soret band was compatible with conversion of the heme iron from its native low spin state to a high spin state with axial symmetry (g ϳ 6.0). Soret band bleaching, due to direct attack on the heme group, was only detected under conditions that favored free radical production (MB ؉ dimer in SDS micelles) or in the presence of a less structured form of the protein (above pH 9.3). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the heme group and the polypeptide chain of cytochrome c with Soret band at 405 nm (cytc405) revealed no alterations in the mass of the cytc405 heme group but oxidative modifications on methionine (Met 65 and Met 80 ) and tyrosine (Tyr 74 ) residues. Damage of cytc405 tyrosine residue impaired its reduction by diphenylacetaldehyde, but not by ␤-mercaptoethanol, which was able to reduce cytc405, generating cytochrome c Fe(II) in the high spin state (spin 2).

The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.

Fluorescence quenching of lipid-bound pyrene was used to assess the binding of cytochrome c (cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM) POPC/DOPE/TOCL, with the conditions that it did or did not contain oxidized phosphatidylcholine molecules, i.e., 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), or a mixture of two hydroperoxide isomers derived from POPC (POPCOX). The binding isotherms reveal two dissociation constants, K(D)(1) and K(D)(2), representing, respectively, the low- and high-affinity states of the membrane. These dissociation constants probably are due to the lipid reorganization promoted by cyt c, as observed in giant unilamellar vesicles that contain fluorescent cardiolipin (CL). The presence of PazePC, which has a nonreactive carboxylic group, increased the K(D)(1) and K(D)(2) values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the K(D)(1) value 1.5-fold, increased the K(D)(2) value 10-fold, and significantly reduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt c incubated with liposomes containing POPCox demonstrated a mass increase corresponding to the formation of nonenal adducts as hydrophobic anchors. Electronic absorption spectroscopy, circular dichroism, and magnetic circular dichroism demonstrated that all of the lipids studied promoted changes in the cyt c coordination sphere. Therefore, in the presence of CL, the oxidation of zwitterionic lipids also promotes changes in the cyt c structure and in the affinity for lipid bilayers.

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