scholarly journals Modulation of ligand–heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond–second temporal range

FEBS Journal ◽  
2013 ◽  
Vol 280 (23) ◽  
pp. 6070-6082 ◽  
Author(s):  
Henry J. Russell ◽  
Samantha J. O. Hardman ◽  
Derren J. Heyes ◽  
Michael A. Hough ◽  
Gregory M. Greetham ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Binding Pocket ◽  
Heme Reactivity

scholarly journals Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts

1998 ◽  
Vol 329 (1) ◽  
pp. 95-99 ◽  
Author(s):  
B. Mark HAMPTON ◽  
Boris ZHIVOTOVSKY ◽  
F. G. Andrew SLATER ◽  
H. David BURGESS ◽  
Sten ORRENIUS
Keyword(s):  
Cytochrome C ◽  
Electrostatic Interactions ◽  
Ionic Composition ◽  
Binding Pocket ◽  
Caspase Activation ◽  
Binding Partner ◽  
Allosteric Interactions ◽  
Transport Chain ◽  
Intracellular Signal ◽  
Chain Interaction

The export of cytochrome c from mitochondria to the cytoplasm has been detected during apoptosis. Addition of cytochrome c to cytosolic extracts can activate the caspases, suggesting that this export could be an important intracellular signal for initiating the apoptotic programme. We have investigated the mechanism of caspase activation by cytochrome c. Mitochondrial cytochrome c normally shuttles electrons between complexes III and IV of the electron transport chain. Interaction with these complexes is dependent on electrostatic interactions via a polylysine binding pocket. Cytosolic caspase activation was only observed with intact holocytochrome c, and increasing the ionic composition of the extracts prevented activation, suggesting that stringent allosteric interactions between cytochrome c and other cytoplasmic factors are necessary. Cytochrome c was fully reduced within 5 min of addition to the cytosolic extracts. Potassium ferricyanide could maintain cytochrome c in an oxidized state, but care was taken to use ferricyanide at concentrations where its polyanion effect did not cause interference. The oxidized form of cytochrome c was able to activate the caspases. We conclude that reduced cytochrome c will function in the cytoplasm; however, its reduction is not a critical step, and electron transfer from cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis.

Nucleic Acid Molecules Prepared by Monolayer Techniques

1972 ◽  
Vol 30 ◽  
pp. 178-179
Author(s):  
Dimitrij Lang
Keyword(s):  
Electron Microscopy ◽  
Standard Deviation ◽  
Nucleic Acid ◽  
Cytochrome C ◽  
Nucleic Acids ◽  
Contour Length ◽  
Sample Standard Deviation ◽  
Molecular Weights ◽  
Length Measurements ◽  
Protein Monolayer

The success of the protein monolayer technique for electron microscopy of individual DNA molecules is based on the prevention of aggregation and orientation of the molecules during drying on specimen grids. DNA adsorbs first to a surface-denatured, insoluble cytochrome c monolayer which is then transferred to grids, without major distortion, by touching. Fig. 1 shows three basic procedures which, modified or not, permit the study of various important properties of nucleic acids, either in concert with other methods or exclusively:1) Molecular weights relative to DNA standards as well as number distributions of molecular weights can be obtained from contour length measurements with a sample standard deviation between 1 and 4%.

Activation of cytochrome c to a peroxidase compound I-type intermediate by H2O2: relevance to redox signalling in apoptosis

2004 ◽  
Vol 71 ◽  
pp. 97-106 ◽  
Author(s):  
Mark Burkitt ◽  
Clare Jones ◽  
Andrew Lawrence ◽  
Peter Wardman
Keyword(s):  
Cytochrome C ◽  
Hydroxyl Radical ◽  
Redox Regulation ◽  
Chemotherapeutic Agents ◽  
Tyrosyl Radical ◽  
Compound I ◽  
Definitive Evidence ◽  
Enhanced Production ◽  
Physico Chemical

The release of cytochrome c from mitochondria during apoptosis results in the enhanced production of superoxide radicals, which are converted to H2O2 by Mn-superoxide dismutase. We have been concerned with the role of cytochrome c/H2O2 in the induction of oxidative stress during apoptosis. Our initial studies showed that cytochrome c is a potent catalyst of 2′,7′-dichlorofluorescin oxidation, thereby explaining the increased rate of production of the fluorophore 2′,7′-dichlorofluorescein in apoptotic cells. Although it has been speculated that the oxidizing species may be a ferryl-haem intermediate, no definitive evidence for the formation of such a species has been reported. Alternatively, it is possible that the hydroxyl radical may be generated, as seen in the reaction of certain iron chelates with H2O2. By examining the effects of radical scavengers on 2′,7′-dichlorofluorescin oxidation by cytochrome c/H2O2, together with complementary EPR studies, we have demonstrated that the hydroxyl radical is not generated. Our findings point, instead, to the formation of a peroxidase compound I species, with one oxidizing equivalent present as an oxo-ferryl haem intermediate and the other as the tyrosyl radical identified by Barr and colleagues [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503]. Studies with spin traps indicated that the oxo-ferryl haem is the active oxidant. These findings provide a physico-chemical basis for the redox changes that occur during apoptosis. Excessive changes (possibly catalysed by cytochrome c) may have implications for the redox regulation of cell death, including the sensitivity of tumour cells to chemotherapeutic agents.

Solution structure of cytochrome b5 mutant (E44/48/56A/D60A) and its interaction with cytochrome c

2001 ◽  
Vol 268 (6) ◽  
pp. 1620-1630
Author(s):  
Yibing Wu ◽  
Yunhua Wang ◽  
Chengmin Qian ◽  
Jun Lu ◽  
Ercheng Li ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Solution Structure ◽  
Cytochrome B5

Expression of sunflower cytochrome c mRNA is tissue-specific and controlled by nitrate and light

1997 ◽  
Vol 99 (2) ◽  
pp. 342-347 ◽  
Author(s):  
Silvina A. Felitti ◽  
Raquel L. Chan ◽  
Gabriela Gago ◽  
Estela M. Valle ◽  
Daniel H. Gonzalez
Keyword(s):  
Cytochrome C ◽  
Tissue Specific

A STUDY OF CYTOCHROME c AND CYTOCHROME b2 CORE IN THE OXIDIZED AND REDUCED STATES USING EXAFS AND XANES SPECTROSCOPIES

1986 ◽  
Vol 47 (C8) ◽  
pp. C8-1143-C8-1146
Author(s):  
I. ASCONE ◽  
J. GOULON ◽  
A. BIANCONI ◽  
M. TEGONI ◽  
F. LABEYRIE
Keyword(s):  
Cytochrome C

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

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