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.

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.

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.

Structure-function relationships in human cytochrome c: The role of tyrosine 67.

Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe-Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH7.0, located in the heme distal pocket, which may act as an acid to cleave the O-O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)-OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals.

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.

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.

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine.

Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding energy values ranging from -7.6 to -10.6 kcal/mol. In agreement with this hypothesis, inhibition studies indicate that chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of Ki being 0.10 µM and 0.55 µM for acetylpolyamine oxidase and spermine oxidase, respectively.

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.

Misfolded proteins and neurodegeneration: role of non-native cytochrome c in cell death.

Intermediates play a relevant role in the protein-folding process, because the onset of diseases of genetic nature is usually coupled with protein misfolding and the formation of stable intermediate species. This article describes and briefly discusses the mechanisms considered responsible, at molecular level, for a number of neurodegenerative diseases. In particular, interest is focused on the newly discovered role of cytochrome c in programmed cell death (apoptosis), consisting of acquisition of powerful cardiolipin-specific peroxidase action. Cardiolipin oxidation induces cytochrome c detachment from the mitochondrial membrane and favors the accumulation of products releasing proapoptotic factors. Cytochrome c showing peroxidase activity has non-native structure, and shows enhanced access of the heme catalytic site to small molecules, such as H(2)O(2). The strict correlation linking cytochrome c with the onset of neurodegenerative disorders is described and the therapeutic approach discussed.

ATP acts as a regulatory effector in modulating structural transitions of cytochrome c: implications for apoptotic activity.

The binding of lipids (free fatty acids as well as acidic phospholipids) to cytochrome c (cyt c) induces conformational changes and partial unfolding of the protein, strongly influencing cyt c oxidase/peroxidase activity. ATP is unique among the nucleotides in being able to turn non-native states of cyt c back to the native conformation. The peroxidase activity acquired by lipid-bound cyt c turns out to be very critical in the early stages of apoptosis. Nucleotide specificity is observed for apoptosome formation and caspase activation, the cleavage occurring only in the presence of dATP or ATP. In this study, we demonstrate the connection between peroxidase activity and oleic acid-induced conformational transitions of cyt c and show how ATP is capable of modulating such interplay. By NMR measurement, we have demonstrated that ATP interacts with a site (S1) formed by K88, R91, and E62 and such interaction was weakened by mutation of E62, suggesting the selective role in the interaction played by the base moiety. Interestingly, the interactions of ATP and GTP with cyt c are significantly different at low nucleotide concentrations, with GTP being less effective in perturbing the S1 site and in eliciting apoptotic activity. To gain insights into the structural features of cyt c required for its pro-apoptotic activity and to demonstrate a regulatory role for ATP (compared to the effect of GTP), we have performed experiments on cell lysates by using cyt c proteins mutated on amino acid residues that, as suggested by NMR measurements, belong to S1. Thus, we provide evidence that ATP acts as an allosteric effector, regulating structural transitions among different conformations and different oxidation states of cyt c, which are endowed with apoptotic activity or not. On this basis, we suggest a previously unrecognized role for ATP binding to cyt c at low millimolar concentrations in the cytosol, beyond the known regulatory role during the oxidative phosphorylation in mitochondria.

Insights into cytochrome c-cardiolipin interaction. Role played by ionic strength.

The finding that cytochrome c (cyt c) plays a role in programmed cell death after its release from the mitochondrion has recently renewed interest in this protein. The structural changes in cytochrome c observed at early stages of the apoptotic process have been related to changes occurring in the protein when it forms a complex with phospholipid vesicles. Among the lipids constituting the membrane, cardiolipin is the one thought to bind to cyt c. In this paper, we have investigated the influence exerted by ionic strength on cytochrome c-cardiolipin interaction and found that formation of the cytochrome c-cardiolipin complex occurs via two distinct transitions, implying a high-affinity site and a low-affinity site. Ionic strength significantly influences complex stability; sodium chloride dissociates the complex through two distinct transitions, the second of which occurs at a very high anion concentration. ATP also dissociates the complex, but under the conditions that were investigated, its action is limited to the high-affinity site. The dissociation process is characterized by a very slow kinetic rate constant ( k obs = 4.2 x 10 (-3) s (-1)) and requires several minutes to be completed. We ascribe it to the high activation barrier met by the protein when restoring the native Fe(III)-M80 axial bond. The peroxidase activity shown by cardiolipin-bound cytochrome c is indicative of a less packed protein tertiary conformation in the complex. In line with earlier reports, these data highlight the manifold functions of cytochrome c besides the well-known role it plays in oxidative phosphorylation, shedding more light on the properties of the cytochrome c-cardiolipin complex, involved in the progression of early stages of apoptosis.

Probing the effect of mutations on cytochrome C stability.

Although the tertiary structures of mitochondrial cytochromes c (cyts c) seem to be remarkably similar, there are variations in their amino acid sequences, stability and functional properties. GdnHCl-induced unfolding experiments on engineered yeast and horse cyt c were carried out with the aim to to clarify, at molecular level, some aspects concerning the stability of this class of proteins. The results obtained are discussed in the light of the three-dimensional structures of the two proteins.

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.

The 40s Omega-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome c.

The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1-38 heme-containing N-fragment with the residues 57-104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega (Omega)-loop region. The fragment complex shows compact structure, native-like alpha-helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1-38/57-104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (p K(a1)=6.72) and a high p K(a)-associated state transition (p K(a2)=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (p K(a)=8.9). The data indicate that the 40s Omega-loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.

Effect of axial coordination on the kinetics of assembly and folding of the two halves of horse heart cytochrome C.

The kinetics of the assembly of two complementary fragments of oxidized horse heart cytochrome c (cyt c), namely the heme-containing fragment-(1-56) and the fragment-(57-104), have been characterized at different pH values. At neutral pH the fragment-(1-56) is hexacoordinated and has two histidines axially ligated to the heme-Fe(III) (Santucci, R., Fiorucci, L., Sinibaldi, F., Polizio, F., Desideri, A., and Ascoli, F. (2000) Arch. Biochem. Biophys. 379, 331-336), thus mimicking what occurs in the folding intermediate of cyt c. The kinetics of the formation of the complex between the two fragments are characterized at pH 7.0 by a slow rate constant that is independent of the concentration of the reactants; conversely, at a low pH the kinetics are much faster and depend on the concentration of the fragments. This behavior suggests that the rate-limiting step observed in the recombination process of the fragments at neutral pH (that leads to the final coordination of Met-80) has to be ascribed to the detachment of the "misligated" histidine. Thus, the faster recombination rate at a low pH can be related to the fact that histidine is protonated and not able to coordinate to the metal. Furthermore, the independence of the rate constant on the concentration of the reactants observed at pH 7.0 can be accounted for by the occurrence of a conformational transition, which takes place immediately after the two fragments collapse together, likely simulating what induces the detachment of the misligated histidine during cytochrome folding.

A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment.

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.

Bovine tryptases. cDNA cloning, tissue specific expression and characterization of the lung isoform.

A complementary DNA encoding a new bovine tryptase isoform (here named BLT) was cloned and sequenced from lung tissue. Analysis of sequence indicates the presence of a 26-amino acid prepro-sequence and a 245 amino acid catalytic domain. It contains six different residues when compared with the previously characterized tryptase from bovine liver capsule (BLCT), with the most significant difference residing at the primary specificity S1 pocket. In BLT, the canonical residues Asp-Ser are present at positions 188-189, while in BLCT these positions are occupied by residues Asn-Phe. This finding was confirmed by mass fingerprinting of the peptide mixture obtained upon in-gel tryptic digestion of BLT. Analysis by gel filtration of the purified protein shows that BLT is probably tetrameric, similar to the previously identified tryptases from other species, with monomer migrating as 35-40 kDa multiple bands in SDS/PAGE. As expected, the catalytic abilities of the two bovine tryptases are different. The specificity constant values (kcat/Km) assayed with model substrates are 10- to 60-fold higher in the case of BLT. The tissue-specific expression of the two tryptases was evaluated at the RNA level by analysis of their different restriction patterns. In lung, only BLT was found to be expressed, while in liver capsule only BLCT is present. Both isoforms are distributed in similar amounts in heart and spleen. Analysis of the two gene sequences reveals the presence of several recognition sequences in the promoter regions and suggest a role for hormones in governing the mechanism of tissue expression of bovine tryptases.