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 Photochemical action spectra indicate that cytochrome a/a3 is the predominant haemoprotein terminal oxidase in Acanthamoeba castellanii

1983 ◽  
Vol 210 (3) ◽  
pp. 721-725 ◽  
Author(s):  
R I Scott ◽  
D Lloyd
Keyword(s):  
Cytochrome B ◽  
Room Temperature ◽  
Stationary Phases ◽  
Acanthamoeba Castellanii ◽  
Terminal Oxidase ◽  
Difference Spectra ◽  
Intact Cells ◽  
Action Spectra ◽  
Stages Of Growth ◽  
Photochemical Action

1. Room-temperature CO-reduced minus reduced difference spectra of intact cells of Acanthamoeba castellanii show the presence of CO-reacting haemoproteins in cells from the early-exponential, late-exponential and stationary phases of growth. 2. The relative rates of reaction with CO of the two haemoproteins differ; that of cytochrome a/a3 with CO is complete within 1 min of bubbling with CO, whereas that of cytochrome b takes longer than 90 min. 3. Photochemical action spectra reveal cytochrome a/a3 as the predominant haemoprotein oxidase at all stages of growth. 4. It is concluded that the alternative oxidases known to be present in these organisms are not cytochromes.

Aerobic and anaerobic respiratory systems in Campylobacter fetus subsp. jejuni grown in atmospheres containing hydrogen

1982 ◽  
Vol 152 (1) ◽  
pp. 306-314
Author(s):  
G M Carlone ◽  
J Lascelles
Keyword(s):  
Cytochrome C ◽  
Cytochrome B ◽  
System Analysis ◽  
Maximum Growth ◽  
Oxidoreductase Activity ◽  
Intact Cells ◽  
Cytochrome O ◽  
Campylobacter Fetus ◽  
Fetus Subsp ◽  
Carbon Monoxide Binding

Maximum growth of Campylobacter fetus subsp. jejuni, strain C-61, occurred when the cultures were incubated with shaking in atmospheres containing approximately 30% hydrogen, 5% oxygen, and 10% CO2. Suspensions of cells grown under these conditions consumed oxygen with formate as the substrate in the presence of 0.33 mM cyanide, which completely inhibited respiration with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine and with lactate. Spectroscopic evidence with intact cells suggested that a form of cytochrome c, reducible with formate but not with lactate or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine, can be reoxidized by a cyanide-insensitive system. Analysis of membranes from the cells showed high- and low-potential forms of cytochrome c, cytochrome b, and various enzymes, including hydrogenase, formate dehydrogenase, and fumarate reductase. The predominant carbon monoxide-binding pigment appeared to be a form of cytochrome c, but the spectra also showed evidence of cytochrome o. The membrane cytochromes were reduced by hydrogen in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide at concentrations which prevented the reduction of cytochrome c with succinate as the electron donor. Reoxidation of the substrate-reduced cytochromes by oxygen was apparently mediated by cyanide-sensitive and cyanide-insensitive systems. The membranes also had hydrogen-fumarate oxidoreductase activity mediated by cytochrome b. We conclude that C. fetus jejuni has high- and low-potential forms of cytochrome which are associated with a complex terminal oxidase system.

scholarly journals Carbon monoxide- and oxygen-reacting haemoproteins in the mitochondrial fraction from the soil amoeba Acanthamoeba castellanii. Studies at subzero temperatures

1981 ◽  
Vol 200 (2) ◽  
pp. 337-342 ◽  
Author(s):  
D Lloyd ◽  
S W Edwards ◽  
B Chance
Keyword(s):  
Carbon Monoxide ◽  
Electron Transport ◽  
Cytochrome C ◽  
Acanthamoeba Castellanii ◽  
Mitochondrial Fraction ◽  
Subzero Temperatures ◽  
Soil Amoeba ◽  
Reduced Forms

1. Mitochondria-enriched fractions of the soil amoeba Acanthamoeba castellanii contained four haemoproteins that in their reduced forms reacted with CO to give photodissociable CO complexes; these were cytochromes a 3, a 614, b- and c-type cytochromes. 2. Non-photodissociable oxygen-containing compounds were formed at temperatures between -130 and -150 degrees C after photodissociation of CO in the presence of 200 microM-O2, 3. Electron transport, indicated by the oxidation of cytochromes a + a3 and cytochrome c, did not occur until the temperature was raised to -80 degrees C.

scholarly journals Stimulation of the respiratory chain of rat liver mitochondria between cytochrome c1 and cytochrome c by glucagon treatment of rats

1978 ◽  
Vol 172 (3) ◽  
pp. 399-405 ◽  
Author(s):  
Andrew P. Halestrap
Keyword(s):  
Cytochrome C ◽  
Cytochrome B ◽  
Respiratory Chain ◽  
Redox State ◽  
Electron Flow ◽  
Rat Liver Mitochondria ◽  
O2 Uptake ◽  
Pyruvate Metabolism ◽  
Cytochromes C ◽  
Cytochrome C1

Mitochondria from glucagon-treated rats oxidize succinate, but not ascorbate plus tetramethylphenylenediamine, faster in the uncoupled state than do control mitochondria. The rate of O2 uptake in the presence of both substrates is equal to the sum of the rates of the O2 uptake in the presence of either substrate alone. It is concluded that the mitochondrial respiratory chain is limited at some point between cytochromes b and c and that this step is regulated by glucagon. Measurement of the cytochrome spectra under uncoupled conditions in the presence of succinate and rotenone demonstrates a crossover between cytochromes c and c1 when control mitochondria are compared with those from glucagon-treated rats, cytochrome c being more oxidized and cytochrome c1 more reduced in control mitochondria. Under conditions where pyruvate metabolism is studied the control mitochondria are generally more oxidized than those from glucagon-treated rats, the redox state of cytochrome b-566 correlating with the rate of pyruvate metabolism in sucrose medium. However, when the redox state of the mitochondria is taken into account, a crossover between cytochromes c and c1 is again apparent. The spectra of the b cytochromes are complex, but cytochrome b-562 appears to become more reduced relative to cytochrome b-566 in mitochondria from glucagon-treated rats than in control mitochondria. This can be explained by the existence of a more alkaline matrix in glucagon-treated rats, the redox potential for cytochrome b being pH-sensitive. It is concluded that glucagon stimulates electron flow between cytochromes c1 and c. The physiological significance of these findings is discussed.

Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase.

1998 ◽  
Vol 201 (8) ◽  
pp. 1129-1139 ◽  
Author(s):  
E Gnaiger ◽  
B Lassnig ◽  
A Kuznetsov ◽  
G Rieger ◽  
R Margreiter
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiratory Chain ◽  
Oxygen Supply ◽  
Oxygen Affinity ◽  
Active State ◽  
Model Systems ◽  
Control Analysis ◽  
Excess Capacity ◽  
Flux Control

The oxygen affinity of the enzyme system involved in mitochondrial respiration indicates, in relation to intracellular oxygen levels and interpreted with the aid of flux control analysis, a significant role of oxygen supply in limiting maximum exercise. This implies that the flux control coefficient of mitochondria is not excessively high, based on a capacity of mitochondrial oxygen consumption that is slightly higher than the capacity for oxygen supply through the respiratory cascade. Close matching of the capacities and distribution of flux control is consistent with the concept of symmorphosis. Within the respiratory chain, however, the large excess capacity of cytochrome c oxidase, COX, appears to be inconsistent with the economic design of the respiratory cascade. To address this apparent discrepancy, we used three model systems: cultured endothelial cells and mitochondria isolated from heart and liver. Intracellular oxygen gradients increase with oxygen flux, explaining part of the observed decrease in oxygen affinity with increasing metabolic rate in cells. In addition, mitochondrial oxygen affinities decrease from the resting to the active state. The oxygen affinity in the active ADP-stimulated state is higher in mitochondria from heart than in those from liver, in direct relationship to the higher excess capacity of COX in heart. This yields, in turn, a lower turnover rate of COX even at maximum flux through the respiratory chain, which is necessary to prevent a large decrease in oxygen affinity in the active state. Up-regulation of oxygen affinity provides a functional explanation of the excess capacity of COX. The concept of symmorphosis, a matching of capacities in the respiratory cascade, is therefore complemented by 'synkinetic' considerations on optimum enzyme ratios in the respiratory chain. Accordingly, enzymatic capacities are matched in terms of optimum ratios, rather than equal levels, to meet the specific kinetic and thermodynamic demands set by the low-oxygen environment in the cell.

scholarly journals The role of succinate in the respiratory chain of Trypanosoma brucei procyclic trypomastigotes

1989 ◽  
Vol 259 (2) ◽  
pp. 363-368 ◽  
Author(s):  
J F Turrens
Keyword(s):  
Cytochrome C ◽  
Trypanosoma Brucei ◽  
Respiratory Chain ◽  
Intact Cell ◽  
Inhibitory Effect ◽  
Fumarate Reductase ◽  
Glycerol Phosphate ◽  
Intact Cells ◽  
Fluorescence Studies ◽  
Nadh Cytochrome

Trypanosoma brucei procyclic trypomastigotes were made permeable by using digitonin (0-70 micrograms/mg of protein). This procedure allowed exposure of coupled mitochondria to different substrates. Only succinate and glycerol phosphate (but not NADH-dependent substrates) were capable of stimulating oxygen consumption. Fluorescence studies on intact cells indicated that addition of succinate stimulates NAD(P)H oxidation, contrary to what happens in mammalian mitochondria. Addition of malonate, an inhibitor of succinate dehydrogenase, stimulated NAD(P)H reduction. Malonate also inhibited intact-cell respiration and motility, both of which were restored by further addition of succinate. Experiments carried out with isolated mitochondrial membranes showed that, although the electron transfer from succinate to cytochrome c was inhibitable by antimycin, NADH-cytochrome c reductase was antimycin-insensitive. We postulate that the NADH-ubiquinone segment of the respiratory chain is replaced by NADH-fumarate reductase, which reoxidizes the mitochondrial NADH and in turn generates succinate for the respiratory chain. This hypothesis is further supported by the inhibitory effect on cell growth and respiration of 3-methoxyphenylacetic acid, an inhibitor of the NADH-fumarate reductase of T. brucei.

scholarly journals Cytochrome c Oxidase Subunit 4 Isoform Exchange Results in Modulation of Oxygen Affinity

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 443 ◽  
Author(s):  
David Pajuelo Reguera ◽  
Kristýna Čunátová ◽  
Marek Vrbacký ◽  
Alena Pecinová ◽  
Josef Houštěk ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Affinity ◽  
Intact Cells ◽  
Knock Out ◽  
Atp Production ◽  
Oxygen Kinetics ◽  
Biochemical Mechanisms

Cytochrome c oxidase (COX) is regulated through tissue-, development- or environment-controlled expression of subunit isoforms. The COX4 subunit is thought to optimize respiratory chain function according to oxygen-controlled expression of its isoforms COX4i1 and COX4i2. However, biochemical mechanisms of regulation by the two variants are only partly understood. We created an HEK293-based knock-out cellular model devoid of both isoforms (COX4i1/2 KO). Subsequent knock-in of COX4i1 or COX4i2 generated cells with exclusive expression of respective isoform. Both isoforms complemented the respiratory defect of COX4i1/2 KO. The content, composition, and incorporation of COX into supercomplexes were comparable in COX4i1- and COX4i2-expressing cells. Also, COX activity, cytochrome c affinity, and respiratory rates were undistinguishable in cells expressing either isoform. Analysis of energy metabolism and the redox state in intact cells uncovered modestly increased preference for mitochondrial ATP production, consistent with the increased NADH pool oxidation and lower ROS in COX4i2-expressing cells in normoxia. Most remarkable changes were uncovered in COX oxygen kinetics. The p50 (partial pressure of oxygen at half-maximal respiration) was increased twofold in COX4i2 versus COX4i1 cells, indicating decreased oxygen affinity of the COX4i2-containing enzyme. Our finding supports the key role of the COX4i2-containing enzyme in hypoxia-sensing pathways of energy metabolism.

scholarly journals Quantitative Analysis of Mitochondrial Ca2+ Uptake and Release Pathways in Sympathetic Neurons

2000 ◽  
Vol 115 (3) ◽  
pp. 371-388 ◽  
Author(s):  
Stephen L. Colegrove ◽  
Meredith A. Albrecht ◽  
David D. Friel
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Sympathetic Neurons ◽  
Differential Sensitivity ◽  
Rate Equations ◽  
Intact Cells ◽  
Isolated Mitochondria ◽  
Cellular Context ◽  
The Impact ◽  
Uptake And Release

Rate equations for mitochondrial Ca2+ uptake and release and plasma membrane Ca2+ transport were determined from the measured fluxes in the preceding study and incorporated into a model of Ca2+ dynamics. It was asked if the measured fluxes are sufficient to account for the [Ca2+]i recovery kinetics after depolarization-evoked [Ca2+]i elevations. Ca2+ transport across the plasma membrane was described by a parallel extrusion/leak system, while the rates of mitochondrial Ca2+ uptake and release were represented using equations like those describing Ca2+ transport by isolated mitochondria. Taken together, these rate descriptions account very well for the time course of recovery after [Ca2+]i elevations evoked by weak and strong depolarization and their differential sensitivity to FCCP, CGP 37157, and [Na+]i. The model also leads to three general conclusions about mitochondrial Ca2+ transport in intact cells: (1) mitochondria are expected to accumulate Ca2+ even in response to stimuli that raise [Ca2+]i only slightly above resting levels; (2) there are two qualitatively different stimulus regimes that parallel the buffering and non-buffering modes of Ca2+ transport by isolated mitochondria that have been described previously; (3) the impact of mitochondrial Ca2+ transport on intracellular calcium dynamics is strongly influenced by nonmitochondrial Ca2+ transport; in particular, the magnitude of the prolonged [Ca2+]i elevation that occurs during the plateau phase of recovery is related to the Ca2+ set-point described in studies of isolated mitochondria, but is a property of mitochondrial Ca2+ transport in a cellular context. Finally, the model resolves the paradoxical finding that stimulus-induced [Ca2+]i elevations as small as ∼300 nM increase intramitochondrial total Ca2+ concentration, but the steady [Ca2+]i elevations evoked by such stimuli are not influenced by FCCP.

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