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1.
The reported X-ray structure of horse-heart ferricytochrome c has been refined by conformational energy calculations, using a three-stage computational procedure. In stage I, the atomic positions are adjusted to conform to idealized bond lengths and bond angles characteristic of small amino acid derivatives, while yet remaining as close as possible to the X-ray coordinates. In stage II, atomic overlaps are eliminated by adjusting the backbone and side-chain dihedral angles to minimize the nonbonded energy, hydrogen-bonded energy, and rotational energy contributions. In the final stage of refinement, the electrostatic energy and a more accurate hydrogen-bonded energy treatment are considered, in addition to the energy contributions of stage II. A "fitting potential" of gradually decreasing strength is imposed in both stages II and III, in order to keep the computed structure as similar to the x-ray structure as is consistent with a low-energy conformation. The final computed structure of cytochrome c exhibits a very low conformational energy (-504 kcal/mol) and also closely resembles the X-ray structure (RMS deviation = 0.77 A for all atoms). However, a special treatment was required in order to alter the location of the phenyl ring of phenylalanine-82. In contrast to the originally published X-ray structure, which shows the phenyl ring pointing away from the heme, the phenyl ring in the computed structure is tucked into the heme crevice, in a position similar to that observed in the reduced form of tuna cytochrome c, in the oxidized form of Rhodospirillum rubrum cytochrome c2, and also in the recently determined structure of oxidized tuna cytochrome c. 相似文献
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A P Boswell G R Moore R J Williams D E Harris C J Wallace S Bocieck D Welti 《The Biochemical journal》1983,213(3):679-686
1H-n.m.r. and 13C-n.m.r. spectroscopy of horse cytochrome c and 1H-n.m.r. spectroscopy of the lysine-modified proteins N epsilon-acetimidyl-, N epsilon-amidino-, N epsilon-trifluoroacetyl- and N epsilon-maleyl-cytochrome c have shown that, although the lysine modifications do not greatly perturb the protein structure at pH7 and 27 degrees C, at higher temperature or at alkaline pH some parts of the structure are markedly perturbed. At pH7 and 27 degrees C the region of the protein about Ile-57 is affected in all the modified proteins, though not all to the same degree. N epsilon-Maleylation most seriously affects the protein structure, and the fully maleylated protein is readily unfolded. At 27 degrees C all four of the tyrosine residues of native horse cytochrome c have pKa values above 11, but in N epsilon-acetimidyl-cytochrome c the pKa of one tyrosine residue is 10.2. 相似文献
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1. The effects of cyanogen bromide on horse-heart cytochrome c and horse-heart myoglobin have been investigated. Cytochrome c yielded four fragments, of which two were haemopeptides. The two colourless peptides had amino acid compositions corresponding to those that are expected, on the basis of the sequence proposed for horse-heart cytochrome c by Margoliash, Smith, Kreil & Tuppy (1961), from cleavage at both methionine residues. Of the two haemopeptides, one was isolated and shown to be that derived from cleavage at only one methionine residue, that nearer to the C-terminus of the peptide chain. 2. Myoglobin also gave four peptides, three of which accounted for the total amino acid content of the intact protein. The fourth fragment arose by cleavage at a single methionine residue, that nearer the C-terminus. Characterization of this fourth fragment made it possible to deduce the order of arrangement of the fragments in the intact molecule. 相似文献
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Spectrophotometric titration of human serum albumin indicates that ionization of the 18 tyrosine residues takes place between pH 9 and 12.7. A Hill plot indicates that protons dissociate co-operatively from tyrosine residues, in pure albumin between pH 11.0 and 11.4 with a Hill coefficient 1.7, and in the bilirubin-albumin complex between pH 11.2 and 11.7 with a Hill coefficient 1.6. With a stopped-flow technique it is shown that about seven of the tyrosines ionize fast, with rate constants well above 10(2) s-1, when pH is suddenly changed from near neutral to pH 11.76. Further residues ionize slowly, with rate constants around 10(2) s-1 or less. The N-form of albumin (pH 6) contains one more fast ionizing tyrosine than the B-form of albumin (pH 10). Binding of bilirubin or laurate to the albumin molecule (molar ratio 1:1) transforms one to three of the fast ionizing tyrosines to slowly ionizing. 相似文献
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E Stellwagen 《Biochemistry》1968,7(7):2496-2501
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The binding of ferricytochrome c to liposomes consisting of phosphatidylcholine mixtures with cardiolipin (3:1) or phosphatidylserine (3:1) has been investigated. Experimental data have been analyzed in terms of two-dimensional models of large ligand adsorption. The equilibrium parameters of ferricytochrome c interaction with a phospholipid bilayer are determined. 相似文献
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S. Hirota H. Okumura S. Kuroiwa N. Funasaki Y. Watanabe 《Journal of biological inorganic chemistry》2005,10(4):355-363
Cytochrome c (cyt c) was reduced by a tyrosine-containing peptide, tyrosyltyrosylphenylalanine (TyrTyrPhe), at pH 6.0–8.0, while tyrosinol or tyrosyltyrosine (TyrTyr) could not reduce cyt c effectively under the same condition. Cyt c was reduced at high peptide concentration, whereas the reaction did not occur effectively at low concentration. The reaction rate varied with time owing to a decrease in the TyrTyrPhe concentration and the production of tyrosine derivatives during the reaction. The initial rate constants were 2.4×10–4 and 8.1×10–4 s–1 at pH 7.0 and 8.0, respectively, for the reaction with 1.0 mM TyrTyrPhe in 10 mM phosphate buffer at 15°C. The reciprocal initial rate constant (1/kint) increased linearly against the reciprocal peptide concentration and against the linear proton concentration, whereas logkint decreased linearly against the root of the ionic strength. These results show that deprotonated (TyrTyrPhe)–, presumably deprotonated at a tyrosine site, reduces cyt c by formation of an electrostatic complex. No significant difference in the reaction rate was observed between the reaction under nitrogen and oxygen atmospheres. From the matrix-assisted laser desorption ionization time-of-flight mass spectra of the reaction products, formation of a quinone and other tyrosine derivatives of the peptide was supported. These products should have been produced from a tyrosyl radical. We interpret the results that a cyt cox/(TyrTyrPhe)–cyt cred/(TyrTyrPhe) equilibrium is formed, which is usually shifted to the left. This equilibrium may shift to the right by reaction of the produced tyrosyl radical with the tyrosine sites of unreacted TyrTyrPhe peptides. 相似文献
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C G Eley G R Moore R J Williams W Neupert P J Boon H H Brinkhof R J Nivard G I Tesser 《The Biochemical journal》1982,205(1):153-165
The tertiary structures of horse, tuna, Neurospora crassa, horse [Hse65,Leu67]- and horse [Hse65,Leu74]-cytochromes c were studied with high-resolution 1H n.m.r. spectroscopy. The amino acid sequences of these proteins differ at position 46, which is occupied by phenylalanine in the horse proteins but by tyrosine in the remaining two, and at positions 67, 74 and 97, which are all occupied by tyrosine residues in horse and tuna cytochrome c but in the other proteins are substituted by phenylalanine or leucine, though there is only one such substitution per protein. The various aromatic-amino-acid substitutions do not seriously affect the protein structure. 相似文献
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The temperature dependence of the nuclear magnetic resonance spectrum of horse ferricytochrome c is described. The protein maintains an ordered structure over the temperature range 20 degrees C to 77 degrees C. The temperature dependence of the spectrum of ferricytochrome c arises from a number of causes including the paramagnetism of the ferric ion and protein structural changes. Preliminary analysis of the data show that the region of the protein about Ile-57 is flexible. Comparison of the data with the analogous data for horse ferrocytochrome c reveals that there is a small difference in structure between cytochrome c in its two oxidation states in the region about Ile-57. 相似文献
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Proton resonance assignments of horse ferricytochrome c 总被引:7,自引:0,他引:7
Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments. 相似文献
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Interaction between sodium dodecyl sulfate and ferricytochrome c 总被引:4,自引:0,他引:4
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The influence of chemical modification of His residues in Mb on the rate of redox reaction in system MbO2--Cyt c has been studied at different ionic strengths and pH medium. The products of alkylation of all available His by bromacetate and iodacetamide, CM-Mb and CA-Mb, respectively, and myoglobin, modified by spin label 2,2', 6,6'-tetramethyl-4-bromoacetoxypiperidine-1-oxyl (SL) at His residue A10--Sl (His-A10)--Mb have been studied. It has been shown, that the character of the ionic strength dependence of reaction SL(His-A10)--MbO2 with Cyt c at pH 6.0 ann 7.0 is basically analogues to that, observed for intact protein. It means that only His-GH1 of two His residues, His-A10 and His-GH1, situated in the region of "active contact" of Mg with Cyt c molecule, participates in the interactions, essential for electron transfer. The interaction of the charge of this His with the negatively charged group of Cyt c is necessary, probably for the proper arrangement of other interactions in the active complex, because the deprotonation of His-GHl in the studied pH interval decreases the rate of the process by more than one order of magnitude. The rate of oxidation of MC-MbO2 and CA-MbO2 by ferricytochrome c, in contrast to intact protein, shows a weak dependence on the ionic strength and does not depend on the pH medium, throughout the range of ionic strengths from 0.005 to 1.0. The cause of the radical change in the ionic strength dependence is, probably, nearly entire disturbance of electrostatic interactions in the active complex due to chemical modification of His residues in the site of "active contact", and first of all, the His-CHl residue. The fact, that during alkylation of all available His in Mb the electron transfer persists in the system, points to that in the process of electron transfer to cytochrome c, uncharged group, most probably "inner" His-B5, participates. Based on the data on spatial structure and the obtained results, the positions of the charged groups in the site of "active contact" of Mb with Cyt c molecule are presented. 相似文献