Glycerol-induced formation of the molten globule from acid-denatured cytochrome c: implication for hierarchical folding

At high concentration (98% or higher, v/v), glycerol induces collapse of acid-denatured cytochrome c into a compact state, the G_U state, showing a molten globule character. The G_U state possesses a nativelike -helix structure but a tertiary conformation less packed with respect to the native state. The spectroscopic properties of the G_U state closely resemble those of the molten globule stabilized by the organic solvent from the native protein (called the G_N state), indicating that glycerol can stabilize the molten globule of cytochrome c either from the native or the acid-denatured protein. The G_U and the G_N states show spectroscopic (and, thus, structural) properties and stabilities comparable to those of molten globules stabilized by different effectors, despite the fact that the mechanisms involved in the molten globule formation may significantly differ. This implies in cytochrome c a hierarchy for the rupture (native-to-molten globule) or the formation (unfolded-to-molten globule) of intramolecular interactions leading to the stabilization of the molten globule state of the protein, independently from the effector responsible for the structural transition, in accord with the sequential model proposed by Englander and collaborators.     

Electrochemical evidence for the molten globule states of cytochrome c induced by N-alkyl sulfates at low concentrations

The molten globule state (MG) of cytochrome c is the major intermediate of protein folding. The formation of MG state of cytochrome c is induced by n-alkyl sulfates such as sodium octyl sulfate (SOS), sodium dodecyl sulfate (SDS), and sodium tetradecyl sulfate (STS). The folding state of cytochrome c was monitored using circular dichroism (CD), isothermal titration calorimetry (ITC) and partial specific volumes. To explore a new approach for characterizing the MG conformation, cyclic voltametric studies of n-alkyl sulfates induced transition at acidic pH of cytochrome c (unfolded state, U) was carried out. Here, we have used a cystein-modified gold electrode, which is effective for direct rapid electron transfer to cytochrome c even in acid solutions, to directly observe electrochemistry in native (N) cytochrome c. Our results show that the extent of electron transfer is increased for UMG, and also the easiness of electron transferring occurred from MGN transition. Thus we demonstrate that the MG state of cytochrome c, induced by n-alkyl sulfates as salts with hydrophobic chains (hydrophobic salts), with different compactness reaches to near identical amount of electron transferring as N state.     

Equilibrium titrations of acid-induced unfolding-refolding and salt-induced molten globule of cytochrome c by FT-IR spectroscopy

Despite extensive investigations on the acid-unfolded and acid/salt-induced molten globule(-like) states of cytochrome c using variety of techniques, structural features of the acid-unfolded state in terms of residual secondary structures and the structural transition between the acid-unfolded and acid/salt-refolded states have not been fully characterized beyond the circular dichroism (CD) spectroscopy. It is unusual that secondary structure(s) of the unfolded state leading to the molten globule state, an important protein folding intermediate, as determined by CD was not fully corroborated by independent experimental method(s). In this study, we carried out an equilibrium titration of acid-induced unfolding and subsequent acid- and salt-induced refolding of cytochrome c using Fourier transform infrared spectroscopy. The spectral profiles of the equilibrium titration reveal new structural details about the acid-unfolded state and the structural transition associated with the acid/salt-refolded molten globule(-like) states of cytochrome c.     

Insights into the role of the histidines in the structure and stability of cytochrome c

In this paper we investigate the role played by each histidine in the amino acid sequence of yeast iso-1-cytochrome c (with the exception of H18, the residue axially coordinated to the heme iron) in determining the protein structure and stability. To this end, we have generated and characterized the double mutants H26Y/H33Y, H26Y/H39K and H33Y/H39K obtained from the C102T variant of the protein, which retain only one histidine side chain in the amino acid sequence. In particular, the H39K mutation inserts a lysine at position 39 as in the sequence of equine cytochrome c. The H26Y/H33Y/H39K triple mutant, which lacks all three histidines, was also produced and its spectroscopic properties are compared with those of the double mutants. The data highlight the critical role played by H26 in determining protein stability. Recombinant horse cytochrome c and the corresponding H26Y mutant were also generated and characterized. Since equine cytochrome c exhibits higher stability than the yeast protein, this provides a valuable opportunity to understand the role played by the invariant H26 residue in determining structure and stability.     

A structural model for the adduct between cytochrome c and cytochrome c oxidase

An ensemble of structural models of the adduct between cytochrome c and cytochrome c oxidase from Paracoccus denitrificans has been calculated based on the experimental data from site-directed mutagenesis and NMR experiments that have accumulated over the last years of research on this system. The residues from each protein that are at the protein–protein interface have been identified by the above experimental work, and this information has been converted in a series of restraints explicitly used in calculations. It is found that a single static structural model cannot satisfy all experimental data simultaneously. Therefore, it is proposed that the adduct exists as a dynamic ensemble of different orientations in equilibrium, and may be represented by a combination or average of the various limiting conformations calculated here. The equilibrium involves both conformations that are competent for electron transfer and conformations that are not. Long-range recognition of the partners is driven by non-specific electrostatic interactions, while at shorter distances hydrophobic contacts tune the reciprocal orientation. Electron transfer from cytochrome bc ₁ to cytochrome c oxidase is mediated through cytochrome c experiencing multiple encounters with both of its partners, only part of which are productive. The number of encounters, and thus the electron transfer rate, may be increased by the formation of a cytochrome bc ₁–cytochrome c oxidase supercomplex and/or (in human) by increasing the concentration of the two enzymes in the membrane space. Protein Data Bank Accession numbers The coordinates of the five best structural models for each of the four clusters have been deposited in the Protein Data Bank (PDB ID 1ZYY).     

Formation of a molten globule like state in bovine serum albumin at alkaline pH

Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26–32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6 M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.     

Structural and thermodynamic behavior of cytochrome c assembled with glutathione-covered gold nanoparticles

The conformational changes of horse heart ferricytochrome c (cyt c) after association of gold nanoparticles have been studied by electronic absorption spectroscopy and circular dichroism (CD). Our results show that the structural stability around the heme of complexed cyt c was increased successfully. Glutathione-layered gold nanoparticles caused a significant increase of the apparent pK values of the cyt c alkaline transition. Similarly, the heme crevice became more stable to heat after assembly of cyt c with gold nanoparticles. In contrast, gold nanoparticles weaken the overall thermal stability of the cyt c by decreasing the denaturation temperature estimated from far-UV CD measurements. Similar behavior has previously been reported for cyt c complexed with physiological redox partners as well as hydrophilic polyanions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The intramitochondrial location of cytochrome c peroxidase in wild-type and petite Saccharomyces cerevisiae

Cytochemical and ultrastructural analysis of wild-type cells of Saccharomyces cerevisiac, grown aerobically in a glucose-limited chemostat, shows that cytochrome c peroxidase is localized between the membranes of the cristae, that is, in the intracristal space. This enzyme is thus positioned appropriately within the organelle to act as an alternate terminal oxidase for the respiratory chain. The proximity of the peroxidase to major sites of generation of its two substrates may account for the small leakage of hydrogen peroxide from yeast mitochondria, as compared with the larger outflow from mammalian mitochondria.In the cytoplasmic petite mutant, gross distortion of promitochondrial membrane arrangement is evident. Nevertheless, cytochrome c peroxidase activity is present in the same amounts as is found in wildtype cell, and is localized predominantly within annuli of membrane which constitute the promitochondria in these cells.No unequivocal evidence was obtained for the localization of catalase in microbodies or other organelles in either wild-type or petite cells.     

Influence of NaCl and sorbitol on the stability of conformations of cytochrome c

Influence of ionic (NaCl) and non-ionic (sorbitol) additives on structural transitions of cytochrome c was investigated by circular dichroism, optical and EPR spectroscopy. Transformations of cytochrome c, induced by the acidification of solution and temperature perturbation, were monitored in the heme pocket together with changes in the secondary structure. NaCl and sorbitol exhibited antagonistic effect on the acid-induced transition of the protein. Sorbitol enhanced the stability of native conformation while NaCl destabilized this state. The midpoints of acid-induced transitions in the axial coordination of heme as well as in the secondary structure occurred nearly at the same pH values. However, temperature-induced transitions in the unfolding of the secondary structure were almost coincidental with the cleavage of Met80–Fe bond only in the sorbitol solutions. In the salt solution the Met80–Fe bond was markedly more labile than the secondary structure.     

Bcl-x(S) induces an NGF-inhibitable cytochrome c release

Bcl-x(S), a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-x(S) and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-x(S) induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-x(S) induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-x(S)-induced apoptosis, and that NGF inhibits Bcl-x(S)-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways.     

Salsolinol,a catechol neurotoxin,induces oxidative modification of cytochrome c

Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), an endogenous neurotoxin, is known to perform a role in the pathogenesis of Parkinson’s disease (PD). In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with salsolinol. When cytochrome c was incubated with salsolinol, protein aggregation increased in a dosedependent manner. The formation of carbonyl compounds and the release of iron were obtained in salsolinol- treated cytochrome c. Salsolinol also led to the release of iron from cytochrome c. Reactive oxygen species (ROS) scavengers and iron specific chelator inhibited the salsolinol-mediated cytochrome c modification and carbonyl compound formation. It is suggested that oxidative damage of cytochrome c by salsolinol might induce the increase of iron content in cells, subsequently leading to the deleterious condition which was observed. This mechanism may, in part, provide an explanation for the deterioration of organs under neurodegenerative disorders such as PD. [BMB Reports 2013; 46(2): 119-123]     

Mammalian mitochondrial DNA evolution: A comparison of the cytochrome b and cytochrome c oxidase II genes

The evolution of two mitochondrial genes, cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with special emphasis on the orders Artiodactyla and Rodentia. When analyzed using both maximum parsimony, with either equal or unequal character weighting, and neighbor joining, neither gene performed with a high degree of consistency in terms of the phylogenetic hypotheses supported. The phylogenetic inconsistencies observed for both these genes may be the result of several factors including differences in the rate of nucleotide substitution among particular lineages (especially between orders), base composition bias, transition/transversion bias, differences in codon usage, and different constraints and levels of homoplasy associated with first, second, and third codon positions. We discuss the implications of these findings for the molecular systematics of mammals, especially as they relate to recent hypotheses concerning the polyphyly of the order Rodentia, relationships among the Artiodactyla, and various interordinal relationships.Correspondence to: R.L. Honeycutt     

Galig, a novel cell death gene that encodes a mitochondrial protein promoting cytochrome c release

Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector. Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL. This antagonism was not observed upon co-transfection of Bcl-2 and galig. Galig encodes a mitochondrial-targeted protein named mitogaligin. Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection. Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release. Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Stability and apoptotic activity of recombinant human cytochrome c

An efficient system for producing human cytochrome c variants is important to help us understand the roles of this protein in biological processes relevant to human diseases including apoptosis and oxidative stress. Here, we describe an Escherichia coli expression system for producing recombinant human cytochrome c. We also characterize the structure, stability, and function of the protein and show its utility for studying apoptosis. Yields of greater than 8 mg of pure protein per liter culture were attained. Circular dichroism spectropolarimetry studies show that the secondary and tertiary structures of the human protein are nearly identical to those of the horse protein, but the human protein is more stable than other eukaryotic cytochromes c. Furthermore, recombinant human cytochrome c is capable of inducing caspase-3 activity in a cell-free caspase activation assay. We use data from this assay along with data from the literature to define the apaf-1 binding site on human cytochrome c.     

Temperature, pH, and solvent isotope effects on cytochrome c peroxidase mutant N82A studied by proton NMR

The mutant of baker's yeast cytochrome c peroxidase-CN with Ala82 in place of Asn82, [N82A]CcPCN, exhibits a complex solution behavior featuring dynamic interconversion among three enzyme forms that so far have only been detected by NMR spectroscopy. Proton NMR studies of [N82A]CcPCN reveal resonances from each of the three enzyme forms and show that the interconversion among forms is controlled by the pH, temperature, and isotope composition (H₂O vs. D₂O) of the buffer solution. No evidence for a key hydrogen bond between His52 and heme-coordinated cyanide is found in any of the enzyme forms, indicating that disruption of the extensive distal hydrogen bonding network is the source of this phenomenon.     

Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid that exerts potent anti-cancer effects in vitro and in vivo, but is non toxic to untransformed cells. In our previous study we observed that BetA consistently induced cell death in a broad panel of tumor cell lines. Apoptosis induced by BetA involves activation of caspases, PARP cleavage and DNA fragmentation and was suggested to depend on the mitochondrial pathway. However, conflicting results have been reported with respect to the role of the pro- and anti-apoptotic members of the Bcl-2 family, which are often aberrantly regulated in tumors and thereby confer growth and survival advantages. Here we show that BetA-induced apoptosis critically depends on the release of cytochrome c from the mitochondria and formation of the apoptosome. Nevertheless, over-expression of Bcl-2 or Bcl-XL only provides limited protection against BetA-induced apoptosis. More importantly, Bax/Bak deficient cells are as sensitive to BetA as their wild-type counterparts, suggesting that cytochrome c is released in a non-classical fashion. In agreement, pre-incubation with cyclosporin A indicated a crucial role for the mitochondrial permeability transition pore (PT) in the induction of apoptosis. Our observations therefore indicate that BetA affects mitochondria and induces cytochrome c release directly via PT Pore. This is only temporarily prevented by anti-apoptotic members of the Bcl-2 family, but independent of Bax and Bak. These findings help to explain the remarkable broad efficacy of BetA against tumor cells of different origin and its effect in tumor cells that are resistant to other chemotherapeutic agents.     

Cytochrome c and SDS: a molten globule protein with altered axial ligation

Saccharomices cerevisiae (yeast iso-1) cytochrome c has been investigated in the presence of 100 mM SDS in order to simulate the interaction of cytochrome c with membrane. Under these circumstances, a high spin species with detached methionine axial ligand is observed through NMR, in analogy to findings on the horse heart protein. However, at variance with the latter system, for the yeast protein also a low spin species is detected, which appears to be present with a concentration of about 40% with respect to that of the high spin species. The R(1), R(2), [1H]-15N NOE of backbone amides which are not affected by paramagnetism are homogeneous and allow a simultaneous analysis of the data for the two species. The result is that the rotational correlation time is larger than in water and larger than expected on the basis of viscosity of the SDS-containing solution. This finding suggests interactions of cytochrome c with SDS. Furthermore, it appears that there is subnanosecond backbone mobility, which also accounts for the decreased intensity of NOE cross-peaks and may be associated with equilibria between helical and random coil structure. The dynamic behavior appears to be a common feature of the high spin and low spin species and is consistent with the presence of a molten globule state. The molten globule nature of the protein could account for the presence of the different axial coordination of the heme iron. Such findings are meaningful with respect to the physiology of cytochrome c as electron transfer protein and as promoter of apoptosis.     

Roles of a short connecting disulfide bond in the stability and function of psychrophilic Shewanella violacea cytochrome c 5*

Cys-59 and Cys-62, forming a disulfide bond in the four-residue loop of Shewanella violacea cytochrome c ₅ (SV cytc ₅), contribute to protein stability but not to redox function. These Cys residues were substituted with Ala in SV cytc ₅, and the structural and functional properties of the resulting C59A/C62A variant were determined and compared with those of the wild-type. The variant had similar features to those of the wild-type in absorption, circular dichroic, and paramagnetic ¹H NMR spectra. In addition, the redox potentials of the wild-type and variant were essentially the same, indicating that removal of the disulfide bond from SV cytc ₅ does not affect the redox function generated in the vicinity of heme. However, calorimetric analysis of the wild-type and variant showed that the mutations caused a drastic decrease in the protein stability through enthalpy, but not entropy. Four residues are encompassed by the SV cytc ₅ disulfide bond, which is the shortest one that has been proved to affect protein stability. The protein stability of SV cytc ₅ can be controlled without changing the redox function, providing a new strategy for regulating the stability and function of cytochrome c.