scholarly journals Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite

1998 ◽  
Vol 332 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Martyn A. SHARPE ◽  
Chris E. COOPER
Keyword(s):  
Nitric Oxide ◽  
Cytochrome C ◽  
Cysteine Residue ◽  
Cross Linking ◽  
Nitrosyl Complex ◽  
Mitochondrial Oxygen Consumption ◽  
Irreversible Inhibition ◽  
Ferrocytochrome C ◽  
Ferricytochrome C ◽  
Dissociation Constant Kd

The aerobic reactions of nitric oxide with cytochrome c were analysed. Nitric oxide (NO) reacts with ferrocytochrome c at a rate of 200 M-1 s-1 to form ferricytochrome cand nitroxyl anion (NO-). Ferricytochrome c was detected by optical spectroscopy; NO- was detected by trapping with metmyoglobin (Mb3+) to form the EPR-detectable Mb–nitrosyl complex, and by the formation of dimers in yeast ferrocytochrome cvia cross-linking of the free cysteine residue. The NO- formed subsequently reacted with oxygen to form peroxynitrite, as measured by the oxidation of dihydrorhodamine 123. NO binds to ferricytochrome c to form the ferricytochrome c-NO complex. The on-rate for this reaction is 1.3±0.4×103 M-1·s-1, and the off-rate is 0.087±0.054 s-1. The dissociation constant (Kd)of the complex is 22±7 µM. These reactions of NO with cytochrome c are likely to be relevant to mitochondrial metabolism of NO. Ferricytochrome c can act as a reversible sink for excess NO in the mitochondria. The reduction of NO to NO- by ferrocytochrome cmay play a role in the irreversible inhibition of mitochondrial oxygen consumption by peroxynitrite. It is generally assumed that peroxynitrite would be formed in mitochondria via the reaction of NO with superoxide. The finding that NO- is formed from the reaction of NO and ferrocytochrome c provides a means of producing peroxynitrite in the absence of superoxide, via the reaction of NO- with oxygen.

scholarly journals A quantitative test for superoxide radicals produced in biological systems

1982 ◽  
Vol 203 (3) ◽  
pp. 551-558 ◽  
Author(s):  
H Kuthan ◽  
V Ullrich ◽  
R W Estabrook
Keyword(s):  
Cytochrome C ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Homogeneous System ◽  
Horse Heart Cytochrome ◽  
Ferrocytochrome C ◽  
Ferricytochrome C ◽  
Primary Amino ◽  
Horse Heart Cytochrome C

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH-cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.

scholarly journals The Respiratory Chain of Beetroot Mitochondria

1960 ◽  
Vol 13 (2) ◽  
pp. 109 ◽  
Author(s):  
JT Wiskich ◽  
RK Morton ◽  
RN Robertson
Keyword(s):  
Cytochrome C ◽  
Absorption Band ◽  
Beta Vulgaris ◽  
Respiratory Chain ◽  
Electron Acceptors ◽  
Root Tissue ◽  
Absorption Bands ◽  
Isolated Mitochondria ◽  
Ferrocytochrome C ◽  
Ferricytochrome C

Mitochondria were isolated from root tissue of red beetroot (Beta vulgaris L.) and the components of the respiratory chain for oxidation of succinate and of reduced diphosphopyridine nucleotide (DPNH) were studied. Succinate, DPNH, ferrocytochrome c, and malate were used as substrates, and 2,6-dichlorophenolindophenol, ferricytochrome c, and oxygen as hydrogen (electron) acceptors. DPNH was oxidized without addition of cytochrome c and malate without addition of DPN. These observations suggest that the respiratory chain was retained intact in the isolated mitochondria. Cytochromes b, C1. and c were identified spectroscopically by the positions of their characteristic ex-absorption bands. The very small amount of cytochrome c present may indicate some loss of this component during isolation of the mitochondria. An absorption band near 600 mp' was attributed to cytochromes (a+a3).

scholarly journals 1H-n.m.r. evaluation of the ferricytochrome c-cardiolipin interaction. Effect of superoxide radicals

1990 ◽  
Vol 265 (1) ◽  
pp. 227-232 ◽  
Author(s):  
B Soussi ◽  
A C Bylund-Fellenius ◽  
T Scherstén ◽  
J Ångström
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Native Structure ◽  
Hydrogen Bond Interactions ◽  
Ferrocytochrome C ◽  
Ischaemia And Reperfusion ◽  
Ferricytochrome C ◽  
Induced Changes ◽  
Alkaline Isomerization

The interaction between ferricytochrome c and cardiolipin was investigated by 1H n.m.r. at 270 MHz. From the phospholipid-induced changes of the protein spectral features it is concluded that the first 2 equivalents of cardiolipin cause a conformational change at the lower part of the solvent-exposed haem edge, involving a rearrangement of the hydrogen-bond interactions of propionate 6, thus partly accounting for the lowered redox potential of cytochrome c in the presence of cardiolipin. The increased value for the pK of the alkaline isomerization of ferricytochrome c shows that cardiolipin stabilizes the native structure of the protein, indicating that the oxidized form assumes ferrocytochrome c-like properties. Peroxidation of cardiolipin by superoxide radical ions drastically decreases the protein binding to this phospholipid. The implications of this finding, and the likelihood of the ternary cytochrome c-cardiolipin-cytochrome c oxidase complex, for the binding of cytochrome c to cytochrome c oxidase in vivo, are discussed in relation to peroxidative damage following ischaemia and reperfusion.

scholarly journals Complex-formation between cytochrome c and cytochrome c peroxidase. Equilibrium and titration studies

1971 ◽  
Vol 121 (1) ◽  
pp. 69-82 ◽  
Author(s):  
Eugene Mochan ◽  
P. Nicholls
Keyword(s):  
Cytochrome C ◽  
Complex Formation ◽  
Column Chromatography ◽  
Binding Constants ◽  
Cytochrome C Peroxidase ◽  
Peroxide Compound ◽  
Ferrocytochrome C ◽  
Kinetic Predictions ◽  
Ferricytochrome C ◽  
C Complex

1. Physical studies of complex-formation between cytochrome c and yeast peroxidase are consistent with kinetic predictions that these complexes participate in the catalytic activity of yeast peroxidase towards ferrocytochrome c. Enzyme–ferricytochrome c complexes have been detected both by the analytical ultracentrifuge and by column chromatography, whereas an enzyme–ferrocytochrome c complex was demonstrated by column chromatography. Estimated binding constants obtained from chromatographic experiments were similar to the measured kinetic values. 2. The physicochemical study of the enzyme–ferricytochrome c complex, and an analysis of its spectrum and reactivity, suggest that the conformation and reactivity of neither cytochrome c nor yeast peroxidase are grossly modified in the complex. 3. The peroxide compound of yeast cytochrome c peroxidase was found to have two oxidizing equivalents accessible to cytochrome c but only one readily accessible to ferrocyanide. Several types of peroxide compound, differing in available oxidizing equivalents and in reactivity with cytochrome c, seem to be formed by stoicheiometric amounts of hydrogen peroxide. 4. Fluoride combines not only with free yeast peroxidase but also with peroxidase–peroxide and accelerates the decomposition of the latter compound. The ligand-catalysed decomposition provides evidence for one-electron reduction pathways in yeast peroxidase, and the reversible binding of fluoride casts doubt upon the concept that the peroxidase–peroxide intermediate is any form of peroxide complex. 5. A mechanism for cytochrome c oxidation is proposed involving the successive reaction of two reversibly bound molecules of cytochrome c with oxidizing equivalents associated with the enzyme protein.

scholarly journals Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93.

1992 ◽  
Vol 267 (13) ◽  
pp. 8739-8743
Author(s):  
J.T. Kindt ◽  
A Woods ◽  
B.M. Martin ◽  
R.J. Cotter ◽  
Y Osawa
Keyword(s):  
Cysteine Residue ◽  
Cross Linking ◽  
Human Hemoglobin

Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria

1977 ◽  
Vol 55 (10) ◽  
pp. 1114-1117 ◽  
Author(s):  
Gerrit H. Bomhoff ◽  
Mary Spencer
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Ferrocytochrome C ◽  
Optimum Ph ◽  
Triton X ◽  
Assay Conditions ◽  
Nonionic Detergents

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents Triton X-114 and Triton X-100, from pea cotyledon mitochondria. Optimum assay conditions were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.

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