The oxidized forms of cytochrome oxidase

1968 ◽  
Vol 46 (11) ◽  
pp. 1371-1379 ◽  
Author(s):  
G. R. Williams ◽  
R. Lemberg ◽  
M. E. Cutler
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Dominant Species ◽  
Redox State ◽  
Electron Flow ◽  
Aerobic Oxidation ◽  
The Difference

Ferric cytochrome oxidase (Soret max. 418 mμ) and "oxygenated" cytochrome oxidase (Soret max. 428 mμ) carry the same number of oxidizing equivalents (two per heme a) available for the oxidation of ferrous cytochrome c. "Oxygenated" oxidase can be formed by the aerobic oxidation of a3+ + a32+CO and therefore the difference from ferric cytochrome oxidase resides in the a3 moiety of the enzyme. When reductant is added to ferric oxidase aerobically, a transient formation of a2+a33+ occurs but at later times the dominant species in the steady state is "oxygenated" oxidase. It is suggested that the reason for the inhibition of electron flow between a2+ and a33+ may be either in conformational restraints or in the redox state of the associated copper. No such block is apparent in the reduction of "oxygenated" oxidase.

Control of proteoliposomal cytochrome c oxidase: the partial reactions

1990 ◽  
Vol 68 (9) ◽  
pp. 1135-1141 ◽  
Author(s):  
Peter Nicholls
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome C Oxidase ◽  
Redox State ◽  
Ph Gradient ◽  
Transfer Model ◽  
State Reduction

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith–Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by ΔpH and by ΔΨ can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.Key words: proteoliposomes, cytochrome c, cytochrome oxidase, membrane potential, pH gradient, cytochrome a, electron transfer.

scholarly journals The effect of rate limitation by cytochrome c on the redox state of the ubiquinone pool in reconstituted NADH: cytochrome c reductase

1987 ◽  
Vol 247 (3) ◽  
pp. 657-662 ◽  
Author(s):  
J S Reed ◽  
C I Ragan
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Complex I ◽  
Redox State ◽  
Electron Flow ◽  
State Level ◽  
Steady State Level ◽  
Cytochrome C Reductase ◽  
Quinone Reduction ◽  
Nadh Cytochrome

The kinetic model of Ragan & Cottingham [(1985) Biochim. Biophys. Acta 811, 13-31] for electron transport through a mobile pool of quinone predicts that, under certain conditions, the normal linear dependence of electron flow on the degree of reduction (or oxidation) of the quinone should no longer be found. These conditions can be met by reconstituted NADH: cytochrome c reductase (Complex I-III from bovine heart) when electron flow is rate-limited by a low concentration of cytochrome c. We show that, in such a system, the dependence of activity (varied by inhibition with rotenone) on the steady-state level of quinone reduction is indeed non-linear and very closely accounted for by the theory.

Regulation of cytochrome oxidase redox state during umbilical cord occlusion in preterm fetal sheep

2007 ◽  
Vol 292 (4) ◽  
pp. R1569-R1576 ◽  
Author(s):  
Laura Bennet ◽  
Vincent Roelfsema ◽  
Justin M. Dean ◽  
Guido Wassink ◽  
Gordon G. Power ◽  
...  
Keyword(s):  
Cytochrome Oxidase ◽  
Umbilical Cord ◽  
Redox State ◽  
Cerebral Oxygenation ◽  
Cerebral Metabolism ◽  
Electron Flow ◽  
Initial Response ◽  
Severe Hypoxia ◽  
Fetal Sheep ◽  
Severe Hypotension

The preterm fetus is capable of surviving prolonged periods of severe hypoxia without neural injury for much longer than at term. To evaluate the hypothesis that regulated suppression of brain metabolism contributes to this remarkable tolerance, we assessed changes in the redox state of cytochrome oxidase (CytOx) relative to cerebral heat production, and cytotoxic edema measured using cerebral impedance, during 25 min of complete umbilical cord occlusion or sham occlusion in fetal sheep at 0.7 gestation. Occlusion was followed by rapid, profound reduction in relative cerebral oxygenation and EEG intensity and an immediate increase in oxidized CytOx, indicating a reduction in electron flow down the mitochondrial electron transfer chain. Confirming rapid suppression of cerebral metabolism there was a loss of the temperature difference between parietal cortex and body at a time when carotid blood flow was maintained at control values. As occlusion continued, severe hypotension/hypoperfusion developed, with a further increase in CytOx levels to a plateau between 8 and 13 min and a progressive rise in cerebral impedance. In conclusion, these data strongly suggest active regulation of cerebral metabolism during the initial response to severe hypoxia, which may help to protect the immature brain from injury.

Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria

1977 ◽  
Vol 55 (7) ◽  
pp. 706-713 ◽  
Author(s):  
Lars Chr. Petersen ◽  
Hans Degn ◽  
Peter Nicholls
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Concentration ◽  
Respiration Rate ◽  
Redox State ◽  
Rat Liver Mitochondria ◽  
Excess Oxygen ◽  
Succinate Oxidation ◽  
State Reduction

1. Coupled, cytochrome-c-depleted ('stripped') rat liver mitochondria reducing oxygen in the presence of exogenous cytochrome c, with succinate or ascorbate as substrates, show marked declines in the steady-state reduction of cytochrome c in excess oxygen on addition of uncouplers. Calculated ratios of maximal turnover in the uncoupled state and in the energized state for the cytochrome c oxidase (EC 1.9.3.1) reaction lie between 3 and 6, as obtained with reconstituted oxidase-containing vesicles. The succinate-cytochrome c reductase activity in such mitochondria shows a smaller response to uncoupler than that of the oxidase.2. The respiration rates of uncoupled mitochondria oxidizing ascorbate in the presence of added cytochrome c follow a Michaelis–Menten relationship with respect to oxygen concentration, in accordance with the pattern found previously with the solubilized oxidase. But succinate oxidation tends to give nonlinear concave-upward double-reciprocal plots of respiration rate against oxygen concentration, in accordance with the pattern found previously with intact uncoupled mitochondria.3. From simultaneous measurements of cytochrome c steady-state reduction, respiration rate, and oxygen concentration during succinate oxidation under uncoupled conditions it is found that at full reduction of cytochrome c, apparent Km for oxygen is 0.9 μM and the maximal oxidase (aa3) turnover is 400 s−1 (pH 7.4, 30 °C).4. The redox state of cytochrome c in uncoupled systems reflects a simple steady state; the redox state of cytochrome c in energized systems tends towards an equilibrium condition with the terminal cytochrome a3, whose apparent potential under these conditions is more negative than that of cytochrome c.

scholarly journals Regulation of gluconeogenesis during exposure of young rats to hypoxic conditions

1971 ◽  
Vol 121 (2) ◽  
pp. 169-178 ◽  
Author(s):  
F. J. Ballard
Keyword(s):  
Steady State ◽  
Concentration Ratio ◽  
Redox State ◽  
Adenine Nucleotides ◽  
Redox Potentials ◽  
Air Content ◽  
Hypoxic Conditions ◽  
Air Atmosphere ◽  
Old Rats ◽  
The Difference

1. Two-day-old rats were exposed at constant temperature to atmospheres containing air and nitrogen with the air content varied in steps from 100 to 0%. By using this system of graded hypoxia a comparison was made between rates of gluconeogenesis from lactate, serine and aspartate in the whole animal and the concentrations of several liver metabolites. 2. Gluconeogenesis, expressed as the percentage incorporation of labelled isotope into glucose plus glycogen, proceeds linearly for 30min when the animals are incubated in a normal air atmosphere, but is completely suppressed if the atmosphere is 100% nitrogen. 3. Preincubation of animals for between 5 and 30min under an atmosphere containing 19% air results in the attainment of a new steady state with respect to gluconeogenesis and hepatic concentrations of ATP, ADP, AMP, lactate, pyruvate, β-hydroxybutyrate and acetoacetate. 4. When lactate (100μmol), aspartate (20μmol) or serine (20μmol) was injected, it was shown that the more severe the hypoxia the greater the depression of gluconeogenesis. Under conditions when gluconeogenesis was markedly inhibited there were no changes in the degree of phosphorylation of hepatic adenine nucleotides, but free [NAD+]/[NADH] ratios fell in both cytosol and mitochondrial compartments of the liver cell. 5. Measurements of total liver NAD+ and NADH showed that the concentrations of these nucleotide coenzymes changed less with anoxia, in comparison with the concentration ratio of free coenzymes. 6. Calculations showed that the difference in NAD+–NADH redox potentials between mitochondrial and cytosol compartments increased with the severity of hypoxia. 7. From the constancy of the concentrations of adenine nucleotides it is concluded that liver of hypoxic rats can conserve ATP by lowering the rate of ATP utilization for gluconeogenesis. Gluconeogenesis may be regulated in turn by the changes in mitochondrial and cytosol redox state.

Nitric oxide regulation of mitochondrial oxygen consumption II: molecular mechanism and tissue physiology

2007 ◽  
Vol 292 (6) ◽  
pp. C1993-C2003 ◽  
Author(s):  
Chris E. Cooper ◽  
Cecilia Giulivi
Keyword(s):  
Nitric Oxide ◽  
Oxygen Consumption ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Molecular Mechanism ◽  
Guanylate Cyclase ◽  
Redox State ◽  
Physiological Role ◽  
Metabolic Effects ◽  
Whole Body

Nitric oxide (NO) is an intercellular signaling molecule; among its many and varied roles are the control of blood flow and blood pressure via activation of the heme enzyme, soluble guanylate cyclase. A growing body of evidence suggests that an additional target for NO is the mitochondrial oxygen-consuming heme/copper enzyme, cytochrome c oxidase. This review describes the molecular mechanism of this interaction and the consequences for its likely physiological role. The oxygen reactive site in cytochrome oxidase contains both heme iron ( a3) and copper (CuB) centers. NO inhibits cytochrome oxidase in both an oxygen-competitive (at heme a3) and oxygen-independent (at CuB) manner. Before inhibition of oxygen consumption, changes can be observed in enzyme and substrate (cytochrome c) redox state. Physiological consequences can be mediated either by direct “metabolic” effects on oxygen consumption or via indirect “signaling” effects via mitochondrial redox state changes and free radical production. The detailed kinetics suggest, but do not prove, that cytochrome oxidase can be a target for NO even under circumstances when guanylate cyclase, its primary high affinity target, is not fully activated. In vivo organ and whole body measures of NO synthase inhibition suggest a possible role for NO inhibition of cytochrome oxidase. However, a detailed mapping of NO and oxygen levels, combined with direct measures of cytochrome oxidase/NO binding, in physiology is still awaited.

scholarly journals Steady-state H+/O stoichiometry of liver mitochondria

1981 ◽  
Vol 200 (3) ◽  
pp. 539-546 ◽  
Author(s):  
M K Al-Shawi ◽  
M D Brand
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Consumption Rate ◽  
Initial Rate ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
O2 Consumption ◽  
Ejection Rate ◽  
Novel Method

We have measured the H+/O stoichiometry of rat liver mitochondria respiring in a steady-state, using a novel method. This involves measuring the initial rate of H+ back-flow into mitochondria after respiratory inhibition, with the assumption that this is equal to the steady-state H+-ejection rate. Division by the steady-state O2-consumption rate yields the H+/O ratio. The H+/O values obtained were: 8.3 +/- 1.0 (mean +/- S.E.M.) for 3-hydroxybutyrate: 8.2 +/- 0.7 for glutamate plus malate; 6.0 +/- 0.2 for succinate; 4.1 +/- 0.3 for ascorbate/tetramethylphenylenediamine and 3.0 +/- 0.1 for ascorbate/ferrocyanide. These values correspond to H+/O stoichiometries for electron flow to oxygen from NAD+-linked substrates, succinate and cytochrome c of 8, 6 and 2 (charge/O ratio = 4) respectively.

scholarly journals Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

1985 ◽  
Vol 230 (3) ◽  
pp. 797-805 ◽  
Author(s):  
M G Tordi ◽  
M C Silvestrini ◽  
A Colosimo ◽  
L Tuttobello ◽  
M Brunori
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Steady State ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Binding Sites ◽  
Time Course ◽  
Product Inhibition ◽  
Inactive Form ◽  
Catalytically Active ◽  
The Stability

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a ‘half-hearted’ behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

scholarly journals An investigation by e.p.r. and optical spectroscopy of cytochrome oxidase during turnover

1982 ◽  
Vol 203 (2) ◽  
pp. 483-492 ◽  
Author(s):  
M T Wilson ◽  
P Jensen ◽  
R Aasa ◽  
B G Malmström ◽  
T Vänngård
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Optical Spectroscopy ◽  
High Spin ◽  
Electron Flux ◽  
Turnover Rates ◽  
G 12 ◽  
Radical Signal ◽  
O2 Binding

Cytochrome oxidase (EC 1.9.3.1; ferrocytochrome c:oxygen oxidoreductase) was studied during steady-state by optical and e.p.r. methods. Starting with either the ‘resting’ or the ‘pulsed’ enzyme, oxidase, cytochrome c, ascorbate and O2 were mixed and the reaction monitored optically. Tetramethylphenylenediamine was used as mediator to poise the steady-state to the desired reduction level. After mixing, the reaction was quenched by the used of rapid-freeze techniques. The e.p.r. spectra of samples captured at increasing tetramethylphenylenediamine concentrations (i.e. higher electron flux) show decreasing g = 2 (Cu A) and g = 3 (cytochrome a) signals. No Cu B or g = 6 signals (high-spin cytochrome a3) could be found during the reaction. Also, the signal with peaks at g = 1.69, 1.78 and 5 as well as the g = 12 signal was hardly detectable at higher turnover rates. The only new signal appearing during turnover is a radical signal, which is discussed in terms of a protein radical. Finally, a scheme is presented, proposing a catalytic cycle for cytochrome oxidase with respect to the O2 binding Cu B-cytochrome a3 unit.

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.

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