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1.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2002,1570(1):9-18
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. 相似文献
2.
Satoshi Nagao Mariko Ueda Hisao Osuka Hirofumi Komori Hironari Kamikubo Mikio Kataoka Yoshiki Higuchi Shun Hirota 《PloS one》2015,10(4)
Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c
551 (PA cyt c
551), and Hydrogenobacter thermophilus cytochrome c
552 (HT cyt c
552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c
552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c
551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c
551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins. 相似文献
3.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2018,1866(11):1073-1083
Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0–4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-induced non-native coordination states of cyt c, very likely due to reversible unfolding of the least stable foldons, is pre-requisite for enhanced peroxidase-like activity of cyt c in its compact state. Dynamic replacement of the native sixth coordination bond of methionine-80 by lysines (72, 73, and 79) and partially also by histidines (26 and 33) provides an efficient way how to increase peroxidase-like activity of cyt c without significant conformational change at physiological conditions. 相似文献
4.
《Biophysical journal》2020,118(1):128-137
Cytochrome c (cyt c) is known for its role in the electron transport chain but transitions to a peroxidase-active state upon exposure to oxidative species. The peroxidase activity ultimately results in the release of cyt c into the cytosol for the engagement of apoptosis. The accumulation of oxidative modifications that accompany the onset of the peroxidase function are well-characterized. However, the concurrent structural and conformational transitions of cyt c remain undercharacterized. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry is a protein footprinting technique used to structurally characterize proteins. FPOP coupled with native ion mobility separation shows that exposure to H2O2 results in the accumulation of a compact state of cyt c. Subsequent top-down fragmentation to localize FPOP modifications reveals changes in heme coordination between conformers. A time-resolved functional assay suggests that this compact conformer is peroxidase active. Altogether, combining FPOP, ion mobility separation, and top-down and bottom-up mass spectrometry allows us to discern individual conformations in solution and obtain a better understanding of the conformational ensemble and structural transitions of cyt c as it transitions from a respiratory role to a proapoptotic role. 相似文献
5.
The reduction of horse heart ferricytochrome c in neutral and slightly alkaline solutions follows biphasic kinetics. The obtained results are consistent with the existence of two conformational forms of ferricytochrome c, cyt c and cyt c1. The conversion of cyt c to cyt c1 occurs by deprotonation (pK~7), followed by a slow conformational change in the protein structure. 相似文献
6.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(11):140265
Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c552 forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c5 exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c552 and Pseudomonas aeruginosa (PA) cyt c551 domain-swapped dimers. Positively charged horse cyt c and HT cyt c552 domain swapped during expression in E. coli, whereas negatively charged PA cyt c551 and SV cyt c5 did not. Oligomers were formed during expression in E. coli for HT cyt c552 attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c551 attached to either signal peptide. HT cyt c552 formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c552 by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c551 and SV cyt c5 did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding. 相似文献
7.
Abhishek Mandal Cody?L. Hoop Maria DeLucia Ravindra Kodali Valerian?E. Kagan Jinwoo Ahn Patrick?C.A. van?der?Wel 《Biophysical journal》2015,109(9):1873-1884
The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly 13C,15N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core. 相似文献
8.
Anders C. Raffalt 《Journal of inorganic biochemistry》2009,103(5):717-722
We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c4 by [Co(bipy)3]2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c4 is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c4 for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c4 function. 相似文献
9.
Rui Huang Meng Zhang Freeborn Rwere Lucy Waskell Ayyalusamy Ramamoorthy 《The Journal of biological chemistry》2015,290(8):4843-4855
Cytochrome P450 reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis. 相似文献
10.
The release of cytochrome c (cyt c) from mitochondria is an important early step during cellular apoptosis, however the precise mechanism by which the outer mitochondrial membrane becomes permeable to these proteins is as yet unclear. Inspired by our previous observation of cyt c crossing the membrane barrier of giant unilamellar vesicle model systems, we investigate the interaction of cyt c with cardiolipin (CL)-containing membranes using the innovative droplet bilayer system that permits electrochemical measurements with simultaneous microscopy observation. We find that cyt c can permeabilize CL-containing membranes by induction of lipid pores in a dose-dependent manner, with membrane lysis eventually observed at relatively high (µM) cyt c concentrations due to widespread pore formation in the membrane destabilizing its bilayer structure. Surprisingly, as cyt c concentration is further increased, we find a regime with exceptionally high permeability where a stable membrane barrier is still maintained between droplet compartments. This unusual non-lytic state has a long lifetime (>20 h) and can be reversibly formed by mechanically separating the droplets before reforming the contact area between them. The transitions between behavioural regimes are electrostatically driven, demonstrated by their suppression with increasing ionic concentrations and their dependence on CL composition. While membrane permeability could also be induced by cationic PAMAM dendrimers, the non-lytic, highly permeable membrane state could not be reproduced using these synthetic polymers, indicating that details in the structure of cyt c beyond simply possessing a cationic net charge are important for the emergence of this unconventional membrane state. These unexpected findings may hold significance for the mechanism by which cyt c escapes into the cytosol of cells during apoptosis. 相似文献
11.
Tianlei Ying Fangfang Zhong Jin Xie Yanjiao Feng Zhong-Hua Wang Zhong-Xian Huang Xiangshi Tan 《Journal of bioenergetics and biomembranes》2009,41(3):251-257
Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pKa value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline
transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular
basis for the biological multi-functions of cyt c. 相似文献
12.
We studied the effect of submicromolar concentrations of cytochrome c (cyt c) on the phase behavior of ternary lipid membranes composed of charged dioleoylphosphatidylglycerol, egg sphingomyelin and cholesterol. The protein was found to induce micron-sized domains in membranes belonging to the single-fluid-phase region of the protein-free ternary mixture and, as a result, to expand the region of coexistence of liquid ordered (Lo) and liquid disordered (Ld) phases. Direct observations on individual vesicles revealed that protein adsorption increases the area of Ld domains. Measurements using a fluorescent analog of cyt c showed that the protein preferentially adsorbs onto domains belonging to the Ld phase. The adsorption was quantitatively characterized in terms of partitioning ratios between the Ld and the Lo phases. The protein was also found to induce vesicle leakage even at relatively low concentrations. In eukaryotic cells under normal physiological conditions, cyt c is localized within the intermembrane space of mitochondria. During cell apoptotis, cyt c is released into the cytosol and its adsorption to intracellular membranes may strongly perturb the lipid distribution within these membranes as suggested by our results. 相似文献
13.
Francesco Francia Bahia Khalfaoui-Hassani Pascal Lanciano Francesco Musiani Louis Noodleman Giovanni Venturoli Fevzi Daldal 《BBA》2019,1860(2):167-179
The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe2S2 cluster, cyt c1 and cyt b, forming two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the electron transfer pathways originating from QH2 oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc1 and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Qo site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc1 activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc1. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH2 oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe2S2 cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH2 oxidation at the Qo site of cyt bc1. 相似文献
14.
Uma D. Vempati Xianlin Han Carlos T. Moraes 《The Journal of biological chemistry》2009,284(7):4383-4391
Cytochrome c (cyt c) is a heme-containing protein that
participates in electron transport in the respiratory chain and as a signaling
molecule in the apoptotic cascade. Here we addressed the effect of removing
mammalian cyt c on the integrity of the respiratory complexes in
mammalian cells. Mitochondria from cyt c knockout mouse cells lacked
fully assembled complexes I and IV and had reduced levels of complex III. A
redox-deficient mutant of cyt c was unable to rescue the levels of
complexes I and IV. We found that cyt c is associated with both
complex IV and respiratory supercomplexes, providing a potential mechanism for
the requirement for cyt c in the assembly/stability of complex
IV.The mitochondrial electron transport chain consists of four multisubunit
complexes, namely, NADH-ubiquinone oxidoreductase (complex
I),2
succinate-ubiquinone oxidoreductase (complex II), ubiquinone-cytochrome
c oxidoreductase (complex III), and cytochrome c oxidase
(complex IV, COX). Cytochrome c (cyt c) shuttles electrons
from oxidative phosphorylation complex III to complex IV. Electrons are
transferred from reduced cyt c sequentially to the CuA
site, heme a, heme a3, and CuB
binuclear center in the complex IV before being finally transferred to
molecular oxygen to generate water
(1). Respiratory complexes are
assembled into supercomplexes (also called respirasomes). These contain
complex I bound to dimeric complex III and a variable copy number of complex
IV (2).In Saccharomyces cerevisiae, cyt c is encoded by two
genes: CYC1 and CYC7. Mutagenesis studies in yeast have
shown that cyt c is required for the assembly of COX
(3,
4). In yeast lacking both the
cyt c genes (CYC1 and CYC7), COX assembly was
absent. It was also shown that cyt c is only structurally required
for COX assembly, because a catalytic mutant of cyt c (W65S) was
sufficient to bring about near normal levels of COX. However, because yeast
lacks complex I, they could not analyze the role of cyt c in the
assembly/stability of complex I. Mammals possess two different isoforms of cyt
c encoded on different chromosomes: the somatic (cyt cS)-
and testis (cyt cT)-specific isoforms. In mouse, the cDNAs bear 74%
homology, whereas the proteins possess 86% identity with most dissimilarity in
the C terminus.Cardiolipin (CL) is an anionic phospholipid present almost exclusively in
the mitochondrial membranes and constitutes 25% of its total phospholipids
(5). Work from several
laboratories showed that CL is essential for the membrane anchorage of the
respiratory supercomplexes. CL has two main roles in the mitochondrial
structure and function, namely, stabilization of mitochondrial membranes and
specific interactions with proteins. CL deficiency results in inefficient
energy transformation by oxidative phosphorylation, swelling of mitochondria,
decreased ATP/oxygen ratio, and reduced membrane potential
(6,
7). In accordance, in S.
cerevisiae lacking CL synthase, the supercomplex comprising complexes III
and IV is unstable (8).
Assembly mutants of COX had significantly reduced CL synthase activity,
whereas assembly mutants of respiratory complex III and complex V showed less
inhibition (9). Subsequently,
the proton gradient across the inner mitochondrial membrane was found to be
important for CL formation and that CL synthase was stimulated by alkaline pH
at the matrix side (10). In
this study, we investigated the role of cyt c depletion on CL levels
by examining its content and composition in cyt c null cells.Here we aimed to answer the following questions: What is the role of cyt
c in the assembly and maintenance of the different respiratory
complexes in mammals? Are there changes in the content/composition of lipids
in the cyt c-ablated cells? Analysis of mouse fibroblasts revealed
that cyt c is essential for the assembly/stability of COX, and a
catalytically mutant form of cyt c cannot rescue the COX defect in
the cyt c null cells. CL and triacylglycerols showed significant
differences in the cyt c null cells, both in content and
composition. 相似文献
15.
Hema S. Aluri David C. Simpson Jeremy C. Allegood Ying Hu Karol Szczepanek Scott Gronert Qun Chen Edward J. Lesnefsky 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Cytochrome c (Cyt c) is a mobile component of the electron transport chain (ETC.) which contains a tightly coordinated heme iron. In pathologic settings, a key ligand of the cyt c's heme iron, methionine (Met80), is oxidized allowing cyt c to participate in reactions as a peroxidase with cardiolipin as a target. Myocardial ischemia (ISC) results in ETC. blockade and increased production of reactive oxygen species (ROS). We hypothesized that during ischemia–reperfusion (ISC-REP); ROS generation coupled with electron flow into cyt c would oxidize Met80 and contribute to mitochondrial-mediated ETC. damage.Methods
Mitochondria were incubated with specific substrates and inhibitors to test the contributions of ROS and electron flow into cyt c. Subsequently, cyt c and cardiolipin were analyzed. To test the pathophysiologic relevance, mouse hearts that underwent ISC-REP were tested for methionine oxidation in cyt c.Results
The combination of substrate/inhibitor showed that ROS production and electron flux through cyt c are essential for the oxidation of methionine residues that lead to cardiolipin depletion. The content of cyt c methionine oxidation increases following ISC-REP in the intact heart.Conclusions
Increase in intra-mitochondrial ROS coupled with electron flow into cyt c, oxidizes cyt c followed by depletion of cardiolipin. ISC-REP increases methionine oxidation, supporting that cyt c peroxidase activity can form in the intact heart.General significance
This study identifies a new site in the ETC. that is damaged during cardiac ISC-REP. Generation of a neoperoxidase activity of cyt c favors the formation of a defective ETC. that activates signaling for cell death. 相似文献16.
Sarkkarai Raja Singh Subash Prakash Gurusamy Muneeswaran Seenivasan Rajesh Kaliappan Muthukumar Veerapandy Vasu 《Molecular simulation》2013,39(6):459-467
Protein tyrosine nitration is well-established post-translational modification occurring in a number of diseases, viz. neurodegenerative, cardiovascular diseases, ageing, etc. Tyrosine-67 (Tyr-67) nitration of cytochrome c (cyt c) was observed under oxidative stress affecting its structure and electron transfer properties. Hence, in this study, molecular dynamics (MD) simulations were carried out at room temperature to investigate the structural and conformational changes in the nitrated cyt c's. MD results revealed that the bond between FE (Heme-105) and S (Met-80) considerably weakened, radius of gyration, backbone and Cα root-mean-square deviations decreased and hydrogen bonding increased in the nitrated cyt c's relative to wild type (WT) cyt c. Ramachandran plot analysis revealed that N- and C-terminal helices also affected by nitration at CE2 carbon atom. Furthermore, essential dynamics analysis showed that amplitude of concerted motion decreased in the nitrated cyt c's, perhaps due to the increase in the hydrogen bonding interaction. Taken together, the structural and conformational changes in the active site Tyr-67 nitrated cyt c may have implications in the loss of electron/proton transfer and gain of apoptotic properties. 相似文献
17.
To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-l-cysteine (NAC) on cell viability, hydrogen peroxide (H2O2) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H2O2 and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur. 相似文献
18.
Sinibaldi F Droghetti E Polticelli F Piro MC Di Pierro D Ferri T Smulevich G Santucci R 《Journal of inorganic biochemistry》2011,105(11):1365-1372
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. 相似文献
19.
Hong Ji Tapan K. Das Anne Puustinen Mårten Wikström Denis L. Rousseau 《Journal of inorganic biochemistry》2010,104(3):318-9972
The structural and functional properties of active site mutants of cytochrome c oxidase from Paracoccus denitrificans (PdCcO) were investigated with resonance Raman spectroscopy. Based on the Fe-CO stretching modes and low frequency heme modes, two conformers (α- and β-forms) were identified that are in equilibrium in the enzyme. The α-conformer, which is the dominant species in the wild-type enzyme, has a shorter heme a3 iron-CuB distance and a more distorted heme, as compared to the β-conformer, which has a more relaxed and open distal pocket. In general, the mutations caused a decrease in the population of the α-conformer, which is concomitant with a decreased in the catalytic activity, indicating that the α-conformer is the active form of the enzyme. The data suggest that the native structure of the enzyme is in a delicate balance of intramolecular interactions. We present a model in which the mutations destabilize the α-conformer, with respect to the β-conformer, and raise the activation barrier for the inter-conversion between the two conformers. The accessibility of the two conformers in the conformational space of CcO plausibly plays a critical role in coupling the redox reaction to proton translocation during the catalytic cycle of the enzyme. 相似文献
20.
The conformational transition of horse heart cyt c in the presence of exogenous thiazole is investigated by NMR spectroscopy. Surprisingly, besides the native form and the ligand-bound form, another species (species A) exists at neutral pH. Titration of thiazole at alkaline condition indicates that this form has Lys as the axial ligands and in fact is one of alkaline cyt c conformers. At a high concentration of thiazole, species A has a dominant population at a pH of approximately 7. It is the first time that a pure alkaline conformer of cyt c is obtained at neutral pH. The observed NOEs show that species A maintains the overall protein fold although large structural rearrangements are expected in the distal pocket. Furthermore, the different effect of imidazole, pyridine and thiazole on the alkaline transformation of cyt c is discussed. 相似文献