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 Oxygen affinity of the respiratory chain of Acanthamoeba castellanii

1983 ◽  
Vol 214 (1) ◽  
pp. 47-51 ◽  
Author(s):  
D Lloyd ◽  
H Mellor ◽  
J L Williams
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Cytochrome B ◽  
Respiratory Chain ◽  
Oxygen Affinity ◽  
Acanthamoeba Castellanii ◽  
Intact Cells ◽  
Cytochrome Aa3 ◽  
Isolated Mitochondria ◽  
Soil Amoeba

Apparent Km values for O2 for the soil amoeba Acanthamoeba castellanii determined polarographically and by bioluminescence gave similar values (0.37 and 0.41 microM respectively). Mitochondria oxidizing succinate or NADH in the presence or absence of ADP gave values in the range 0.21-0.36 microM-O2. Oxidation of respiratory-chain components to 50% of the aerobic steady states in intact cells was observed at the following O2 concentrations: cytochrome aa3, 0.1-0.25 microM; cytochrome c, 0.3-0.6 microM; cytochrome b, 0.35-0.45 microM; flavoprotein, 2 microM. In isolated mitochondria corresponding values for a-, c- and b-type cytochromes were 0.007, 0.035-0.05 and 0.06-0.09 microM-O2. It is concluded that an O2 gradient exists between plasma membrane and mitochondria in A. castellanii.

scholarly journals The mechanism of transmembrane ΔμH+ generation in mitochondria by cytochrome c oxidase

1979 ◽  
Vol 182 (1) ◽  
pp. 133-147 ◽  
Author(s):  
M Lorusso ◽  
F Capuano ◽  
D Boffoli ◽  
R Stefanelli ◽  
S Papa
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiratory Chain ◽  
Electron Flow ◽  
Aerobic Oxidation ◽  
Outer Side ◽  
Rat Liver Mitochondria ◽  
Proton Release ◽  
Ferrocytochrome C ◽  
Proton Production

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.

Reactions of superoxide anion, catechols, and cytochrome c

1970 ◽  
Vol 48 (8) ◽  
pp. 935-939 ◽  
Author(s):  
Richard W. Miller
Keyword(s):  
Cytochrome C ◽  
Superoxide Anion ◽  
Catalytic Reduction ◽  
Electron Acceptors ◽  
Dihydroxybenzoic Acid ◽  
Fast Reaction ◽  
Reduction Processes ◽  
Ferrocytochrome C ◽  
Iron Sulfur ◽  
Oxidation Reduction

Photoreduced flavins react very rapidly with molecular oxygen to give the superoxide anion. This radical is highly reactive with cytochrome c and p-nitro blue tetrazolium salts. The reduction of these electron acceptors under aerobic conditions is strongly inhibited by catechols such as 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) and 3,4-dihydroxybenzoic acid. Tiron also is essential for the photosensitized reoxidation of ferrocytochrome c in the presence of flavins and oxygen. These findings support a proposal that catechols react rapidly with superoxide anion to yield a new oxidizing species. This fast reaction effectively prevents the oxygen radical from participating in slower oxidation–reduction processes. Inhibition by Tiron of the oxygen-dependent catalytic reduction of electron acceptors is therefore indicative of the participation of enzymatically generated superoxide anion. The proposed explanation of the observed phenomena does not require interaction of the catechol inhibitor with the iron–sulfur chromophores of superoxide-forming metalloflavoproteins.

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 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 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 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.

PARTICULATE DIHYDROOROTATE OXIDASE SYSTEM FROM A PSEUDOMONAD: LINKAGE WITH THE RESPIRATORY CHAIN

1967 ◽  
Vol 45 (9) ◽  
pp. 1283-1294 ◽  
Author(s):  
Richard W. Miller ◽  
Carolyn T. Kerr
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Respiratory Chain ◽  
Heme Iron ◽  
Primary Site ◽  
Electron Acceptors ◽  
Stopped Flow ◽  
Oxidase System ◽  
Respiratory Inhibitors ◽  
Non Heme Iron

A particulate dihydroorotate oxidase system was prepared from a soil pseudomonad. Components of the respiratory chain participating in electron transport from dihydroorotate to molecular oxygen are bound non-heme iron, ubiquinone, cytochromes b and c, and cytochrome oxidase. Alternate pathways to oxygen are also operative. Inhibition by conventional respiratory inhibitors was incomplete. Dyes and added cytochrome c were readily reduced by dihydroorotate. Pyridine–adenine dinucleotide coenzymes were not reduced by the substrate. However, oxidase activities for these cofactors may have prevented any net reduction. The primary site of reaction with dihydroorotate probably consists of a dehydrogenase which is linked to the respiratory chain and is reactive with various dyes.In the absence of external electron acceptors or inhibitors, 0.5 mole of oxygen was consumed per mole of dihydroorotate oxidized. The anaerobic rate of reduction of bound cytochrome c, as studied by the stopped–flow technique, was slower than the maximum initial rates of orotate production.

First Nano-Infrared Spectroscopy and Imaging Measurements of Single Phospholipid Bilayers

2018 ◽  
Author(s):  
Adrian Cernescu ◽  
Michał Szuwarzyński ◽  
Urszula Kwolek ◽  
Karol Wolski ◽  
Paweł Wydro ◽  
...  
Keyword(s):  
Infrared Spectroscopy ◽  
Ir Spectroscopy ◽  
Absorption Band ◽  
Spectral Region ◽  
Near Field ◽  
Phospholipid Bilayers ◽  
Model Systems ◽  
Absorption Bands ◽  
Protein Complement ◽  
20 Nm

<div><div>Scattering-mode Scanning Near-Field Optical Microscopy (sSNOM) allows one to obtain absorption spectra in the mid-IR region for samples as small as 20 nm in size. This configuration has made it possible to measure FTIR spectra of the protein complement of membranes. (Amenabar 2013) We now show that mid-IR sSNOM has the sensitivity required to measure spectra of phospholipids in individual bilayers in the spectral range 800 cm<sup>-1</sup>–1400 cm<sup>-1</sup>. We have observed the main absorption bands of the dipalmitoylphosphatidylcholine headgroups in this spectral region above noise level. We have also mapped the phosphate absorption band at 1070 cm<sup>-1</sup> simultaneously with the AFM topography. We have shown that we could achieve sufficient contrast to discriminate between single and multiple phospholipid bilayers and other structures, such as liposomes. This work opens the way to further research that uses nano-IR spectroscopy to describe the biochemistry of cell membranes and model systems.</div></div><div></div>

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