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1.
Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b. 相似文献
2.
Yu Hirano Makoto Higuchi Hirozo Oh-oka Zheng-Yu Wang 《Journal of molecular biology》2010,397(5):1175-1187
In green sulfur photosynthetic bacteria, the cytochrome cz (cyt cz) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt cz subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt cz subunit (C-cyt cz) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt cz consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt cz supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt cz. 相似文献
3.
Hwei-Ming Peng Jiayan Liu Sarah E. Forsberg Hong T. Tran Sean M. Anderson Richard J. Auchus 《The Journal of biological chemistry》2014,289(49):33838-33849
Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: 84EVLIKK89-b5: 53EQAGGDATENFEDVGHSTDAR73 and CYP17A1-R347K: 341TPTISDKNR349-b5: 40FLEEHPGGEEVLR52. Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis. 相似文献
4.
Elisabeth C. Lowe Sarah Bydder Robert S. Hartshorne Hannah L. U. Tape Elizabeth J. Dridge Charles M. Debieux Konrad Paszkiewicz Ian Singleton Richard J. Lewis Joanne M. Santini David J. Richardson Clive S. Butler 《The Journal of biological chemistry》2010,285(24):18433-18442
Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (Em + 234 ± 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified (∼24 and ∼6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c4 family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 ± 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c4 is a novel route for a member of the DMSO reductase family of molybdoenzymes. 相似文献
5.
Kouta Takeda Takayuki Uchihashi Hiroki Watanabe Takuya Ishida Kiyohiko Igarashi Nobuhumi Nakamura Hiroyuki Ohno 《PloS one》2015,10(2)
An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (RRMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics. 相似文献
6.
Célia M. Silveira Pedro O. Quintas Isabel Moura José J. G. Moura Peter Hildebrandt M. Gabriela Almeida Smilja Todorovic 《PloS one》2015,10(6)
Cytochrome cd1 nitrite reductases (cd
1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd
1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd
1NiR from Marinobacter hydrocarbonoclasticus (Mhcd
1) depends on the presence of its physiological redox partner, cytochrome c
552 (cyt c
552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd
1 and cyt c
552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd
1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd
1 // cyt c
552 and Ag // SAM // cyt c
552 // Mhcd
1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c
552 retains its native properties, while the redox potential of apparently intact Mhcd
1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd
1, reinforcing the idea that subtle and very specific interactions between Mhcd
1 and cyt c
552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd
1. 相似文献
7.
Djemel Hamdane Chuanwu Xia Sang-Choul Im Haoming Zhang Jung-Ja P. Kim Lucy Waskell 《The Journal of biological chemistry》2009,284(17):11374-11384
NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of
electrons to all known microsomal cytochromes P450. A CYPOR variant, with a
4-amino acid deletion in the hinge connecting the FMN domain to the rest of
the protein, has been crystallized in three remarkably extended conformations.
The variant donates an electron to cytochrome P450 at the same rate as the
wild-type, when provided with sufficient electrons. Nevertheless, it is
defective in its ability to transfer electrons intramolecularly from FAD to
FMN. The three extended CYPOR structures demonstrate that, by pivoting on the
C terminus of the hinge, the FMN domain of the enzyme undergoes a structural
rearrangement that separates it from FAD and exposes the FMN, allowing it to
interact with its redox partners. A similar movement most likely occurs in the
wild-type enzyme in the course of transferring electrons from FAD to its
physiological partner, cytochrome P450. A model of the complex between an open
conformation of CYPOR and cytochrome P450 is presented that satisfies
mutagenesis constraints. Neither lengthening the linker nor mutating its
sequence influenced the activity of CYPOR. It is likely that the analogous
linker in other members of the diflavin family functions in a similar
manner.NADPH-cytochrome P450 oxidoreductase
(CYPOR)4 is a
∼78-kDa, multidomain, microsomal diflavin protein that shuttles electrons
from NADPH → FAD → FMN to members of the ubiquitous cytochrome P450
superfamily (1,
2). In humans, the cytochromes
P450 (cyt P450) are one of the most important families of proteins involved in
the biosynthesis and degradation of a vast number of endogenous compounds and
the detoxification and biodegradation of most foreign compounds. CYPOR also
donates electrons to heme oxygenase
(3), cytochrome
b5 (4), and
cytochrome c (5).The FAD receives a hydride anion from the obligate two electron donor NADPH
and passes the electrons one at a time to FMN. The FMN then donates electrons
to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts
electrons at two different steps in its complex reaction cycle. Ferric cyt
P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the
second of the two electrons to form the peroxo
(Fe+3OO)2- cyt P450 intermediate
(6). In vivo, CYPOR
cycles between the one- and three-electron reduced forms
(7,
8). Although the one-electron
reduced form is an air-stable, neutral blue semiquinone (FMNox/sq,
-110 mV), it is the FMN hydroquinone (FMNsq/hq, -270 mV), not the
semiquinone, that donates an electron to its redox partners
(8–11).
CYPOR is the prototype of the mammalian diflavin-containing enzyme family,
which includes nitric-oxide synthase
(12), methionine synthase
reductase (13,
14), and a novel reductase
expressed in the cytoplasm of certain cancer cells
(15). CYPOR is also a target
for anticancer therapy, because it reductively activates anticancer prodrugs
(16).CYPOR consists of an N-terminal single α-helical transmembrane anchor
(∼6 kDa) responsible for its localization to the endoplasmic reticulum and
the soluble cytosolic portion (∼66 kDa) capable of reducing cytochrome
c. Crystal structures of the soluble form of the wild-type and
several mutant CYPORs are available
(17,
18). The first ∼170 amino
acids of the soluble domain are highly homologous to flavodoxin and bind FMN
(FMN domain), whereas the C-terminal portion of the soluble protein consists
of a FAD- and NADPH-binding domain with sequence and structural similarity to
ferredoxin-NADP+ oxidoreductase (FAD domain). A connecting domain,
possessing a unique sequence and structure, joins the FMN and FAD domains and
is partly responsible for the relative orientation of the FMN and FAD domains.
In the crystal structure, a convex anionic surface surrounds FMN. In the
wild-type crystal structure, the two flavin isoalloxazine rings are in van der
Waals contact, poised for efficient interflavin electron transfer
(17). Based on the
juxtaposition of the two flavins, an extrinsic electron transfer rate of
∼1010 s-1 is predicted
(19). However, the
experimentally observed electron transfer rate between the two flavins is
30–55 s-1
(20,
21). This modest rate and
slowing of electron transfer in a viscous solvent (75% glycerol) suggest that
interflavin electron transfer is likely conformationally gated. Moreover, the
“closed” crystal structure, in which the flavins are in contact,
is difficult to reconcile with mutagenesis studies that indicate the acidic
amino acid residues on the surface near FMN are involved in interacting with
cyt P450 (22). The first
structural insight into how cyt P450 might interact with the FMN domain of
CYPOR was provided by the crystal structure of a complex between the heme and
FMN-containing domains of cyt P450 BM3
(23). In this complex, the
methyl groups of FMN are oriented toward the heme on the proximal surface of
cyt P450 BM3. Considered together, these three observations, the slow
interflavin electron transfer, the mutagenesis data, and the structure of the
complex between the heme and FMN domains of cyt P450 BM3, suggest that CYPOR
will undergo a large conformational rearrangement in the course of shuttling
electrons from NADPH to cyt P450. In addition, crystal structures of various
CYPOR variants indicate that the FMN domain is highly mobile with respect to
the rest of the molecule
(18).Consideration of how the reductase would undergo a reorientation to
interact with its redox partners led us to hypothesize the existence of a
structural element in the reductase that would regulate the conformational
changes and the relative dynamic motion of the domains. Our attention focused
on the hinge region between the FMN and the connecting domain, because it is
often disordered and highly flexible in the crystal structure (supplemental
Fig. S1). The length and sequence of the hinge have been altered by
site-directed mutagenesis, and the effects of the mutations on the catalytic
properties of each mutant have been determined. The results demonstrate that
lengthening the linker or altering its sequence do not modify the properties
of CYPOR. In contrast, deletion of four amino acids markedly disrupts electron
transfer from FAD to FMN, whereas the ability of the FMN domain to donate
electrons to cyt P450 remains intact. The hinge deletion variant has been
crystallized in three “open” conformations capable of interacting
with cyt P450. 相似文献
8.
The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by 1H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand. 相似文献
9.
Andrea L. M. Spencer Ireena Bagai Donald F. Becker Erik R. P. Zuiderweg Stephen W. Ragsdale 《The Journal of biological chemistry》2014,289(43):29836-29858
Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. 相似文献
10.
Valerian E. Kagan Hülya A. Bayır Natalia A. Belikova Olexandr Kapralov Yulia Y. Tyurina Vladimir A. Tyurin Jianfei Jiang Detcho A. Stoyanovsky Peter Wipf Patrick M. Kochanek Joel S. Greenberger Bruce Pitt Anna A. Shvedova Grigory Borisenko 《Free radical biology & medicine》2009,46(11):1439-1453
Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt c’s conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria—its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition—are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented. 相似文献
11.
《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
12.
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13.
Satya P. Panda Wenbing Li Priya Venkatakrishnan Li Chen Andrei V. Astashkin Bettie Sue S. Masters Changjian Feng Linda J. Roman 《FEBS letters》2013
Neuronal nitric oxide synthase μ (nNOSμ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSμ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSμ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSμ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme–nitrosyl complex formation. 相似文献
14.
Cvetelin Vasilev Amanda A. Brindley John D. Olsen Rafael G. Saer J. T. Beatty C. N. Hunter 《Photosynthesis research》2014,119(1-2):169-180
Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c 2, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c 2 molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His12-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c 2 is approximately 480 pN with an interaction frequency of around 66 %. The latter value lowered 5.5-fold when chemically reduced RC-His12-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c 2 to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes. 相似文献
15.
Simanta Sarani Paul Pallabi Sil Shubhasis Haldar Samaresh Mitra Krishnananda Chattopadhyay 《The Journal of biological chemistry》2015,290(23):14476-14490
Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c. 相似文献
16.
In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c2 (cyt c2) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c2 (cyt c2) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c2 is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association. 相似文献
17.
Shun Hirota Masaaki Endo Tomoya Tsukazaki Teruhiro Takabe O. Yamauchi 《Journal of biological inorganic chemistry》1998,3(6):563-569
Interactions of charged peptides, such as aspartic acid peptides (Aspptds) and lysine peptides (Lysptds), with cytochrome
c (cyt c) or plastocyanin (PC) have been studied by measuring electron transfer between [Fe(CN)6]4– and cyt c or PC in the presence of these peptides. Aspptds, up to penta-aspartic acid, served as competitive inhibitors of electron transfer from [Fe(CN)6]4– to oxidized cyt c, while Lysptds, up to penta-lysine, promoted electron transfer from [Fe(CN)6]4– to oxidized PC. The electron transfer inhibitory effects of Aspptds are explained as competitive inhibition due to neutralization
of the positively charged amino acid residues at the surface of cyt c by electrostatic interactions, whereas the electron transfer promoting effects of Lysptds may be due to formation of PC·Lysptd
or Lysptd·[Fe(CN)6]4– complexes subsequently forming an electron transferring complex, PC·Lysptd·[Fe(CN)6]4–, without repulsion of the negative charges. The inhibitory effect of Aspptds and promotional effect of Lysptds became significant
as the net charge or concentration of the peptides increased. The promotional effects of Lysptds decreased as the net charge
of the PC negative patch was decreased by mutagenesis. Thus, charged peptides may serve as a probe for investigation of the
molecular recognition character of proteins.
Received: 19 May 1998 / Accepted: 27 July 1998 相似文献
18.
Oleksandr Kokhan Vladimir P. Shinkarev Colin A. Wraight 《The Journal of biological chemistry》2010,285(29):22513-22521
We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c1 in detergent-solubilized bc1 complex from Rhodobacter sphaeroides. Imidazole binding to cyt c1 substantially lowers the midpoint potential of the heme and fully inhibits bc1 complex activity. Temperature dependences showed that binding of Im (Kd ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH0 = −56 kJ/mol, ΔS0 = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (Kd ≈ 9.3 mm) and is entropically driven (ΔH0 = 47 kJ/mol, ΔS0° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c1 triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c2 suggested that Im binding to cyt c1 is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c2, and c1, which showed hydrogen bonds formed between the NδH of Im and the cyt c1 protein, or with a water molecule sequestered with the ligand in the heme cleft. 相似文献
19.
D. Fernando Estrada Jennifer S. Laurence Emily E. Scott 《The Journal of biological chemistry》2013,288(23):17008-17018
The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site. 相似文献
20.
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