Cytotoxicity and internalization analysis of silicon nanowires in Buffalo Green Monkey cells: a preliminary study to evaluate the possibility of carrying viruses inside the cells.

Silicon nanowires (SiNWs) are attractive functional nanomaterials for biomedical applications. The ability to easily tune their size and density, potential biocompatibility, and knowledge of the chemical activation of SiNWs surface make them natural tools to interact with biological materials. We evaluated the possibility of exploiting SiNWs as carriers to introduce organic compounds into cells. The cellular toxicity and the internalization capacity of free-standing and label-free SiNWs were tested on Buffalo Green Monkey cells (BGM). Confocal fluorescent observation of SiNWs conjugated with fluorescein-polyethylene imine (PEI) confirmed the internalization of the NWs into the Buffalo Green Monkey Cells (BGM).

Cytochrome c: An extreme multifunctional protein with a key role in cell fate.

Cytochrome c, a protein that belongs to class 1 of the c-type cytochrome family, exerts different functions depending on its cellular localization and the conditions in which it operates; therefore, it can be defined as 'extreme multifunctional' protein. It mediates electron-transfer in the respiratory chain and acts as a detoxifying agent to dispose of ROS. In addition, cytochrome c plays a role in cell apoptosis. After its release into the cytosol, the protein binds to APAF-1, activates pro-caspase 9, and triggers an enzymatic cascade leading to cell death. The interaction with cardiolipin, one of the phospholipids making up the mitochondrial membrane, is essential to start apoptosis; the binding partially unfolds cytochrome c, alters the heme pocket region, and facilitates detachment of Met80 from the sixth coordination position of the heme iron. These events change the function of cytochrome c from an electron-transfer shuttle to a peroxidase-like hemoprotein, capable to trigger the process that leads to cell death. This review provides an overview of the key role played by the cytochrome c-cardiolipin interaction in apoptosis. This is not only important per se, it provides interesting perspectives for applications in clinical diagnostics that use the protein as a biomarker.

Nanometric ion pair complexes of tobramycin forming microparticles for the treatment of Pseudomonas aeruginosa infections in cystic fibrosis.

Sustained pulmonary delivery of tobramycin from microparticles composed of drug/polymer nanocomplexes offers several advantages against traditional delivery methods. Namely, in patients with cystic fibrosis, microparticle delivery can protect the tobramycin being delivered from strong mucoadhesive interactions, thus avoiding effects on its diffusion toward the infection site. Polymeric ion-pair complexes were obtained starting from two synthetic polyanions, through impregnation of their solid dissociated forms with tobramycin in aqueous solution. The structure of these polymeric systems was characterized, and their activities were examined against various biofilm-forming Pseudomonas aeruginosa. Once dried, the nanocomplexes can change their aggregation state, to form microparticle-based aggregates with a spherical shape and a micrometer size. In aqueous dispersions, the ion-pair complexes produced had nanometric size, negative ζ potential, and high biocompatibility toward human bronchial epithelium cells. The antibiofilm activity of these formulations was more efficient than for free tobramycin, with the antibiofilm activity against P. aeruginosa mucoid and nonmucoid end-stage strains isolated from cystic fibrosis lungs being of particular relevance.

Potential Role of Cytochrome c and Tryptase in Psoriasis and Psoriatic Arthritis Pathogenesis: Focus on Resistance to Apoptosis and Oxidative Stress.

Psoriasis (PsO) is an autoimmune disease characterized by keratinocyte proliferation, chronic inflammation and mast cell activation. Up to 42% of patients with PsO may present psoriatic arthritis (PsA). PsO and PsA share common pathophysiological mechanisms: keratinocytes and fibroblast-like synoviocytes are resistant to apoptosis: this is one of the mechanism facilitating their hyperplasic growth, and at joint level, the destruction of articular cartilage, and bone erosion and/or proliferation. Several clinical studies regarding diseases characterized by impairment of cell death, either due to apoptosis or necrosis, reported cytochrome c release from the mitochondria into the extracellular space and finally into the circulation. The presence of elevated cytochrome c levels in serum has been demonstrated in diseases as inflammatory arthritis, myocardial infarction and stroke, and liver diseases. Cytochrome c is a signaling molecule essential for apoptotic cell death released from mitochondria to the cytosol allowing the interaction with protease, as the apoptosis protease activation factor, which lead to the activation of factor-1 and procaspase 9. It has been demonstrated that this efflux from the mitochondria is crucial to start the intracellular signaling responsible for apoptosis, then to the activation of the inflammatory process. Another inflammatory marker, the tryptase, a trypsin-like serine protease produced by mast cells, is released during inflammation, leading to the activation of several immune cells through proteinase-activated receptor-2. In this review, we aimed at discussing the role played by cytochrome c and tryptase in PsO and PsA pathogenesis. To this purpose, we searched pathogenetic mechanisms in PUBMED database and review on oxidative stress, cytochrome c and tryptase and their potential role during inflammation in PsO and PsA. To this regard, the cytochrome c release into the extracellular space and tryptase may have a role in skin and joint inflammation.

Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 Activity.

Polyunsaturated fatty acids have been reported to play a protective role in a wide range of diseases characterized by an increased metalloproteinases (MMPs) activity. The recent finding that omega-3 and omega-6 fatty acids exert an anti-inflammatory effect in periodontal diseases has stimulated the present study, designed to determine whether such properties derive from a direct inhibitory action of these compounds on the activity of MMPs. To this issue, we investigated the effect exerted by omega-3 and omega-6 fatty acids on the activity of MMP-2 and MMP-9, two enzymes that actively participate to the destruction of the organic matrix of dentin following demineralization operated by bacteria acids. Data obtained (both in vitro and on ex-vivo teeth) reveal that omega-3 and omega-6 fatty acids inhibit the proteolytic activity of MMP-2 and MMP-9, two enzymes present in dentin. This observation is of interest since it assigns to these compounds a key role as MMPs inhibitors, and stimulates further study to better define their therapeutic potentialities in carious decay.

Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only.

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.

The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket.

The spectroscopic and functional properties of the single Met80Ala and double Tyr67His/Met80Ala mutants of human cytochrome c have been investigated in their ferric and ferrous forms, and in the presence of different ligands, in order to clarify the reciprocal effect of these two residues in regulating the access of exogenous molecules into the heme pocket. In the ferric state, both mutants display an aquo high spin and a low spin species. The latter corresponds to an OH- ligand in Met80Ala but to a His in the double mutant. The existence of these two species is also reflected in the functional behavior of the mutants. The observation that (i) a significant peroxidase activity is present in the Met80Ala mutants, (ii) the substitution of the Tyr67 by His leads to only a slight increase of the peroxidase activity in the Tyr67His/Met80Ala double mutant with respect to wild type, while the Tyr67His mutant behaves as wild type, as previously reported, suggests that the peroxidase activity of cytochrome c is linked to an overall conformational change of the heme pocket and not only to the disappearance of the Fe-Met80 bond. Therefore, in human cytochrome c there is an interplay between the two residues at positions 67 and 80 that affects the conformation of the distal side of the heme pocket, and thus the sixth coordination of the heme.

The key role played by charge in the interaction of cytochrome c with cardiolipin.

Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c-CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys â†’ Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein.

Cyanide binding to ferrous and ferric microperoxidase-11.

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cytc). MP11 is characterized by a covalently linked solvent-exposed heme group, the heme-Fe atom being axially coordinated by a histidyl residue. Here, the reactions of ferrous and ferric MP11 (MP11-Fe(II) and MP11-Fe(III), respectively) with cyanide have been investigated from the kinetic and thermodynamic viewpoints, at pH 7.0 and 20.0 °C. Values of the second-order rate constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 4.5 M(-1) s(-1) and 8.9 × 10(3) M(-1) s(-1), respectively. Values of the first-order rate constant for cyanide dissociation from ligated MP11-Fe(II) and MP11-Fe(III) are 1.8 × 10(-1) s(-1) and 1.5 × 10(-3) s(-1), respectively. Values of the dissociation equilibrium constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 3.7 × 10(-2) and 1.7 × 10(-7) M, respectively, matching very well with those calculated from kinetic parameters so that no intermediate species seem to be involved in the ligand-binding process. The pH-dependence of cyanide binding to MP11-Fe(III) indicates that CN(-) is the only binding species. Present results have been analyzed in parallel with those of several heme-proteins, suggesting that (1) the ligand accessibility to the metal center and cyanide ionization may modulate the formation of heme-Fe-cyanide complexes, and (2) the general polarity of the heme pocket and/or hydrogen bonding of the heme-bound ligand may affect cyanide exit from the protein matrix. Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c. Penta-coordinated MP11 displays a very high reactivity towards cyanide, whereas the reactivity of hexa-coordinated horse heart cytochrome c is very low.

The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin.

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4], whereas the value of k on for NO2 (-) reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M(-1) s(-1) (at pH 7.4). CL facilitates the NO2 (-)-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 (-)-mediated conversion of CL-CM-cytc-Fe(II) to CL-CM-cytc-Fe(II)-NO (5.6 ± 0.6 M(-1) s(-1); at pH 7.4) being slightly higher than that for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO (2.6 ± 0.3 M(-1) s(-1); at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10(-6) M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are -1.05 ± 0.07 and -1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH(-). These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL-CM-cytc.

NO2(-)-mediated nitrosylation of ferrous microperoxidase-11.

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cyt c) and characterized by a covalently-linked solvent-exposed heme group. Here, kinetics of the NO2(-)-mediated nitrosylation of ferrous MP11 (MP11-Fe(II)) is reported. Data were obtained between pH6.4 and 8.2, at 20.0°C. The NO2(-)-mediated conversion of MP11-Fe(II) to MP11-Fe(II)-NO requires one proton; accordingly, values of the apparent second-order rate constant (kon) decrease by about two orders of magnitude from (2.9±0.3)×10(1)M(-1)s(-1) to (5.0±0.6)×10(-1)M(-1)s(-1) upon increasing pH from 6.4 to pH8.2. The slope of the linear fitting of Logkon versus pH is -1.00±0.06. Values of kon for the NO2(-)-mediated nitrosylation of MP11-Fe(II) are similar to those of penta-coordinated cardiolipin-bound horse heart cyt c, exceeding by about two orders of magnitude those of wild-type horse heart cyt c. Present results highlight the role of heme distal residues in modulating horse heart cyt c reactivity.

Cardiolipin-cytochrome c complex: Switching cytochrome c from an electron-transfer shuttle to a myoglobin- and a peroxidase-like heme-protein.

Cytochrome c (cytc) is a small heme-protein located in the space between the inner and the outer membrane of the mitochondrion that transfers electrons from cytc-reductase to cytc-oxidase. The hexa-coordinated heme-Fe atom of cytc displays a very low reactivity toward ligands and does not exhibit significant catalytic properties. However, upon cardiolipin (CL) binding, cytc achieves ligand binding and catalytic properties reminiscent of those of myoglobin and peroxidase. In particular, the peroxidase activity of the cardiolipin-cytochrome c complex (CL-cytc) is critical for the redistribution of CL from the inner to the outer mitochondrial membranes and is essential for the execution and completion of the apoptotic program. On the other hand, the capability of CL-cytc to bind NO and CO and the heme-Fe-based scavenging of reactive nitrogen and oxygen species may affect apoptosis. Here, the ligand binding and catalytic properties of CL-cytc are analyzed in parallel with those of CL-free cytc, myoglobin, and peroxidase to dissect the potential mechanisms of CL in modulating the pro- and anti-apoptotic actions of cytc.

Ferric microperoxidase-11 catalyzes peroxynitrite isomerization.

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c offering the possibility to study the reactivity of the heme group relatively unshielded by the protein. Here, the peroxynitrite isomerization to NO3(-) catalyzed by ferric MP11 (MP11-Fe(III)) is reported. Data were obtained between pH3.6 and 8.1, at 20.0°C. The value of the second-order rate constant (kon) for peroxynitrite isomerization to NO3(-) by MP11-Fe(III) decreases from (1.1±0.1)×10(5)M(-1)s(-1), at pH3.6, to (6.1±0.6)×10(3)M(-1)s(-1), at pH8.1. The pH dependence of kon (pKa=6.9) suggests that peroxynitrous acid reacts preferentially with MP11-Fe(III). The MP11-Fe(III)-catalyzed isomerization of peroxynitrite to NO3(-) has been ascribed to the reactive penta-coordinated heme-Fe atom of MP11-Fe(III). In fact, cyanide binding to the sixth coordination position of the heme-Fe atom inhibits the MP11-Fe(III)-catalyzed isomerization of peroxynitrite to NO3(-). The values of the first-order rate constant (k0) for isomerization of peroxynitrite to NO3(-) in the presence of the MP11-Fe(III)-CN complex are superimposable to those obtained in the absence of MP-Fe(III). Values of kon for peroxynitrite isomerization to NO3(-) by MP11-Fe(III) overlap those obtained for penta-coordinated cardiolipin-cytochrome c complex and for carboxymethylated cytochrome c in absence and presence of cardiolipin. Present results highlight the role of the heme-Fe(III) co-ordination state in the modulation of cytochrome c reactivity.

Reductive nitrosylation of the cardiolipin-ferric cytochrome c complex.

Native horse heart cytochrome c (cytc) displays a very low reactivity toward ligands and does not exhibit catalytic properties. However, upon bovine cardiolipin (CL) binding, cytc achieves myoglobin-like properties. Here, NO binding to CL-cytc(III) between pH 7.2 and 9.5, at 20 °C, is reported. At pH 7.2, CL-cytc(III) undergoes reversible nitrosylation, whereas between pH 7.9 and 9.5 CL-cytc(III) undergoes irreversible reductive nitrosylation leading to the formation of CL-cytc(II)-NO. Over the whole pH range explored, first-order kinetics of NO binding to CL-cytc(III) (k = 9.3 s(-1) ) indicates that ligand binding is limited by the cleavage of the weak heme-Fe distal bond. Between pH 7.9 and 9.5, nitrosylated CL-cytc(III) converts to the ligand-free ferrous derivative (CL-cytc(II)), this process being pH-dependent (hOH- = 3.0 × 10(3) M(-1) s(-1) ). Then, CL-cytc(II) converts to nitrosylated CL-cytc(II), in the presence of NO excess. The value of the second-order rate constant for CL-cytc(II) nitrosylation is essentially pH-independent, the average value of lon being 1.4 × 10(7) M(-1) s(-1) . These results agree with the view that CL-cytc nitrosylation may play a role in apoptosis regulation.

Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c.

Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO2 (-)-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k on for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M(-1) s(-1) (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10(-6) M, (1.8 ± 0.2) × 10(-6) M, and (1.4 ± 0.2) × 10(-6) M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm.

Role of lysines in cytochrome c-cardiolipin interaction.

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

Conversion of cytochrome c into a peroxidase: inhibitory mechanisms and implication for neurodegenerative diseases.

A further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H2O2. To investigate the peroxidase activity of non-native cyt c, different forms of the protein were analyzed with the aim to correlate their structural features with the acquired enzymatic activity and apoptogenic properties (wt cyt c/CL complex and two single cyt c variants, H26Y and Y67H, free and bound to CL). The results suggest that cyt c may respond to different environments by changing its fold thus favouring the exertion of different biological functions in different pathophysiological cell conditions. Transitions among different conformations are regulated by endogenous molecules such as ATP and may be affected by synthetic molecules such as minocycline, thus suggesting a mechanism explaining its use as therapeutic agent impacting on disease-associated oxidative and apoptotic mechanisms.

Peroxynitrite detoxification by horse heart carboxymethylated cytochrome c is allosterically modulated by cardiolipin.

Carboxymethylation of equine heart cytochrome c (cytc) changes its tertiary structure by disrupting the heme-Fe-Met80 distal bond, such that carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) on peroxynitrite isomerization by ferric CM-cytc (CM-cytc-Fe(III)) is reported. Unlike native ferric cytc (cytc-Fe(III)), CM-cytc-Fe(III) catalyzes peroxynitrite isomerization, the value of the second order rate constant (k(on)) is 6.8 × 10(4)M(-1)s(-1). However, CM-cytc-Fe(III) is less effective in peroxynitrite isomerization than CL-bound cytc-Fe(III) (CL-cytc-Fe(III); k(on)=3.2 × 10(5)M(-1)s(-1)). Moreover, CL binding to CM-cytc-Fe(III) facilitates peroxynitrite isomerization (k(on)=5.3 × 10(5)M(-1)s(-1)). Furthermore, the value of the dissociation equilibrium constant for CL binding to CM-cytc-Fe(III) (K=1.8 × 10(-5)M) is lower than that reported for CL-cytc-Fe(III) complex formation (K=5.1 × 10(-5)M). Although CM-cytc-Fe(III) and CL-cytc-Fe(III) display a different heme distal geometry and heme-Fe(III) reactivity, the heme pocket and the CL cleft are allosterically linked.

The effects of ATP and sodium chloride on the cytochrome c-cardiolipin interaction: the contrasting behavior of the horse heart and yeast proteins.

In cells a portion of cytochrome c (cyt c) (15-20%) is tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane. The CL-bound protein, which has nonnative tertiary structure, altered heme pocket, and disrupted Fe(III)-M80 axial bond, is thought to play a role in the apoptotic process. This has attracted considerable interest in order to clarify the mechanisms governing the cyt c-CL interaction. Herein we have investigated the binding reaction of CL with the c-type cytochromes from horse heart and yeast. Although the two proteins possess a similar tertiary architecture, yeast cyt c displays lower stability and, contrary to the equine protein, it does not bind ATP and lacks pro-apoptotic activity. The study has been performed in the absence and in the presence of ATP and NaCl, two compounds that influence the (horse cyt c)-CL binding process and, thus, the pro-apoptotic activity of the protein. The two proteins behave differently: while CL interaction with horse cyt c is strongly influenced by the two effectors, no effect is observed for yeast cyt c. It is noteworthy that NaCl induces dissociation of the (horse cyt c)-CL complex but has no influence on that of yeast cyt c. The differences found for the two proteins highlight that specific structural factors, such as the different local structure conformation of the regions involved in the interactions with either CL or ATP, can significantly affect the behavior of cyt c in its reaction with liposomes and the subsequent pro-apoptotic action of the protein.