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1.
The structure of oxidized Rhodopseudomonas palustris cytochrome c
556 has been modeled after that of high-spin cytochrome c from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals. The aim was to exploit the special restraints arising from the paramagnetism of the metal ion. A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints. A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints. The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated. The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0511-2 相似文献
2.
P. M. Paes de Sousa S. R. Pauleta M. L. Simões Gonçalves G. W. Pettigrew I. Moura M. M. Correia dos Santos J. J. G. Moura 《Journal of biological inorganic chemistry》2007,12(5):691-698
This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies
were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E
0′, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by
pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper
protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 × 105 M−1 s−1 was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9. 相似文献
3.
The reduction thermodynamics of cytochrome c (cytc), determined electrochemically, are found to be sensitive to solvent H/D isotope effects. Reduction of cytochrome c is enthalpically more favored in D2O with respect to H2O, but is disfavored on entropic grounds. This is consistent with a reduction-induced strengthening of the H-bonding network within the hydration sphere of the protein. No significant changes in E° occur, since the above variations are compensative. As a main result, this work shows that the oxidation-state-dependent differences in protein solvation, including electrostatics and solvent reorganization effects, play an important role in determining the individual enthalpy and entropy changes of the reduction process. It is conceivable that this is a common thermodynamic feature of all electron transport metalloproteins. The isotope effects turn out to be sensitive to buffer anions which specifically bind to cytc. Evidence is gained that the solvation thermodynamics of both redox forms of cytc are sensibly affected by strongly hydrated anions. 相似文献
4.
The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction. 相似文献
5.
Payne RB Casalot L Rivere T Terry JH Larsen L Giles BJ Wall JD 《Archives of microbiology》2004,181(6):398-406
Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide. 相似文献
6.
The minimal mono-heme ferricytochrome
c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.Abbreviations Bpcytc
soluble fragment of cytochrome c553 from Bacillus pasteurii
- GdmCl
guanidinium chloride
- I75A
Ile75 to Ala mutant
- I75V
Ile75 to Val mutant
- P72A
Pro72 to Ala mutant
- P72G
Pro72 to Gly mutant
- Q68K
Gln75 to Lys mutant
- WT
wild type 相似文献
7.
Cytochrome c peroxidase (CcP) variants with an engineered Mn(II) binding site, including MnCcP [CcP(MI, G41E, V45E, H181D)], MnCcP(W191F), and MnCcP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200–700 cm–1 and 1300–1650 cm–1 regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of CcP(MI), the recombinant yeast CcP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the 3 mode indicate that a pure five-coordinate heme iron exists in CcP(MI) whereas a six-coordinate low-spin iron is the dominant species in the CcP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants CcP(MI, W191F) and CcP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H2O2 and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnCcP(W191F, W51F).Electronic Supplementary Material Supplementary material is available in the online version of this article at .Abbreviations CcP
cytochrome c peroxidase
- CcP(MI)
recombinant yeast CcP containing Met-Ile at the N-terminus in addition to the normal wild-type CcP sequence
- HRP
horseradish peroxidase
- MnCcP
CcP(MI, G41E, V45E, H181D)
- MnCcP(W191F)
CcP(MI, G41E, V45E, H181D, W191F)
- MnCcP(W191F, W51F)
CcP(MI, G41E, V45E, H181D, W191F, W51F)
- MnP
manganese peroxidase
- RR
resonance Raman
- WtCcP
wild-type cytochrome c peroxidase 相似文献
8.
Smirnova IA Zaslavsky D Fee JA Gennis RB Brzezinski P 《Journal of bioenergetics and biomembranes》2008,40(4):281-287
The ba
3-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa
3–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O2 to water is catalysed at a haem a
3-CuB catalytic site. The three-dimensional structure of the ba
3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial,
Rhodobacter sphaeroides and Paracoccus denitrificans aa
3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba
3
-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence
of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 104 s−1) and formation of the peroxy (PR) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 104 s−1, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s−1, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle
was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the PR and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba
3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa
3-type oxidases.
Smirnova and Zaslavsky contributed equally to the work described in this paper. 相似文献
9.
Many bacteria adapt to microoxic conditions by synthesizing a particular cytochrome c oxidase (cbb
3) complex with a high affinity for O2, encoded by the ccoNOQP operon. A survey of genome databases indicates that ccoNOQP sequences are widespread in all sub-branches of Proteobacteria but otherwise are found only in bacteria of the CFB group (Cytophaga, Flexibacter, Bacteroides). Our analysis of available genome sequences suggests four major strategies of regulating ccoNOQP expression in response to O2. The most widespread strategy involves direct regulation by the O2-responsive protein Fnr. The second strategy involves an O2-insensitive paralogue of Fnr, FixK, whose expression is regulated by the O2-responding FixLJ two-component system. A third strategy of mixed regulation operates in bacteria carrying both fnr and fixLJ-fixK genes. Another, not yet identified, strategy is likely to operate in the -Proteobacteria Helicobacter pylori and Campylobacter jejuni which lack fnr and fixLJ-fixK genes. The FixLJ strategy appears specific for the -subclass of Proteobacteria but is not restricted to rhizobia in which it was originally discovered. 相似文献
10.
Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E(m)(app)) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction. 相似文献
11.
The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae. 相似文献
12.
Naoki?Tachiiri Hikaru?Hemmi Shin-ichi?Joseph?Takayama Hajime?Mita Jun?Hasegawa Yoshihiro?Sambongi Yasuhiko?Yamamoto
The paramagnetic susceptibility () tensors of the oxidized forms of thermophile Hydrogenobacter thermophilus cytochrome c552 (Ht cyt c552) and a quintuple mutant (F7A/V13 M/F34Y/E43Y/V78I; qm) of mesophile Pseudomonas aeruginosa cytochrome c551 (Pa cyt c551) have been determined on the basis of the redox-dependent 1H NMR shift changes of the main-chain NH and CH proton resonances of non-coordinated amino acid residues and the NMR structures of the reduced forms of the corresponding proteins (J. Hasegawa, T. Yoshida, T. Yamazaki, Y. Sambongi, Y. Yu, Y. Igarashi, T. Kodama, K. Yamazaki, Y. Kyogoku, Y. Kobayashi (1998) Biochemistry 37:9641–9649; J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi,Y. Igarashi (2000) J Biol Chem 275:37824–37828). From the tensors determined, we obtained the contact shifts for heme methyl proton resonances, which provided the heme electronic structures of the oxidized forms of Ht cyt c552 and qm. We also characterized the heme electronic structure of the cyanide adducts of the proteins, where the axial Met was replaced by an exogenous cyanide ion, through the analysis of 1H NMR spectra. The results indicated that the heme electronic structures of both the proteins in their oxidized forms with axial His and Met coordination are largely different to each other, while those in their cyanide adducts are similar to each other. These results demonstrated that the orientation of the axial Met sulfur lone pair, with respect to heme, predominantly contributes to the spin delocalization into the porphyrin- system of heme in the oxidized proteins with axial His and Met coordination.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations COSY correlation spectroscopy - DQF-COSY double quantum filtered COSY - TOCSY total correlation spectroscopy - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect correlated spectroscopy - Cyt c cytochrome c - Pa cyt c551 Pseudomonas aeruginosa cytochrome c551 - Ht cyt c552 Hydrogenobacter thermophilus cytochrome c552 - obs observed shift - para paramagnetic shift - dia diamagnetic shift - con contact shift - pc pseudo-contact shift 相似文献
13.
We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan. 相似文献
14.
Cytochromes c were found in the cells of the bacterium Geobacter sulfurreducens AM-1 grown on acetate and methacrylate. The periplasmic extract of G. sulfurreducens AM-1 contained about 88% of the total content of cytochromes c of intact cells. The analysis of cytochromes c from the native cells of G. sulfurreducens AM-1, from the periplasmic extract and from the cells treated by an alkaline solution showed the presence of nine proteins containing heme c. The molecular masses of cytochromes c from G. sulfurreducens AM-1 were 12.5, 15.5, 25.7, 29.5, 34.7, 41.7, 50.1, 63.1, and 67.6 kDa; localization of each cytochrome c was determined. Three heme-containing proteins (15.5 kDa, 25.7 kDa, and 29.5 kDa with the most intensive staining) were present mainly in the periplasm of the bacterium. The other two (50.1 and 67.6 kDa) were supposedly localized in the cell membrane. Cytochromes c with the molecular masses of 12.5, 15.5, and 67.6 kDa are considered as possible components of the methacrylate redox system of G. sulfurreducens AM-1. 相似文献
15.
A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan 相似文献
16.
A pure, active cytochrome b
6
f was isolated from the chloroplasts of the marine green alga, Bryopsis
corticulans. To investigate and characterize this cytochrome b
6
f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b
6 to Cytf was 2.01 : 1. The cytochromeb
6
f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b
6
f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b
6
f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively. 相似文献
17.
L-Lactate cytochrome c oxidoreductase (flavocytochrome b 2, FC b 2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b 2 producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b 2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b 2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b 2 producer characterized by a sixfold increased (to 3 μmol min?1 mg?1 protein in cell-free extract) activity of the enzyme. 相似文献
18.
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
19.
The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C4-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C3 and C4 enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C4 pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C4 pattern expression in this plant. 相似文献
20.
Circular dichroism, nuclear magnetic resonance, electron paramagnetic resonance, UV-vis absorption, and resonance Raman (RR)
spectroscopic techniques were employed to study protein and heme structural changes of cytochrome c (Cyt-c) induced by sodium dodecyl sulfate (SDS) monomers and micelles via hydrophobic and electrostatic interactions, respectively.
Both modes of interactions cause the transition to the conformational state B2, which is implicated to be involved in the
physiological processes of Cyt-c. At sub-micellar concentrations of SDS, specific binding of only ca. three SDS monomers, which is likely to occur at the
hydrophobic peptide segment 81–85, is sufficient for a complete conversion to a B2 state in which Met80 is replaced by His33
(His26). These heme pocket structural changes are not linked to secondary structure changes of the protein brought about by
nonspecific binding of SDS monomers in different regions of the protein. Upon binding of micelles, B2 high-spin species can
also be stabilized by electrostatic interactions. In addition, the micelle interaction domain is located on the front surface
of Cyt-c, which includes a ring-like arrangement of lysine residues appropriate for binding one micelle. According to freeze-quench
RR and stopped-flow experiments, state B2 is formed on the long millisecond timescale and reveals a complex dependence on
the SDS concentration that can be interpreted in terms of competitive binding of monomers and micelles. 相似文献