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 Cytochrome oxidase assembly does not require catalytically active cytochrome c.

2003 ◽  
Vol 278 (43) ◽  
pp. 42728
Author(s):  
Antoni Barrientos ◽  
Danielle Pierre ◽  
Johnson Lee ◽  
Alexander Tzagoloff
Keyword(s):  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Catalytically Active

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.

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.

Time course of muscular atrophy during immobilization of hindlimbs in rats

1977 ◽  
Vol 43 (4) ◽  
pp. 656-661 ◽  
Author(s):  
F. W. Booth
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Exponential Decay ◽  
Time Course ◽  
Citrate Synthase ◽  
Muscular Atrophy ◽  
State Level ◽  
Steady State Level ◽  
Plaster Casts

The hindlimbs of rats were immobilized, in plaster casts, for varying durations, and the time course for atrophy of muscle and of selected proteins in these muscles was determined. In those muscles whose lengths were at less than resting length during the fixation procedures, exponential decay to a new apparent steady state after atrophy was shown by wet and dry muscle weights and by the amounts of biuret protein, cytochrome c, and citrate synthase. The time taken to decrease to one-half of the final decrease at the new apparent steady state level was about 4–6 days for the above parameters which decayed exponentially. In contrast, the myoglobin concentration increased during atrophy and the amount of myoglobin remain unchanged during atrophy. When fixation procedures on limbs were such that muscles were stretched to lengths greater than resting length, then the onset of atrophy was delayed; indeed, in some cases muscles hypertrophied when fixed in the stretched position.

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.

scholarly journals Redox-linked spin-state changes in the di-haem cytochrome c-551 peroxidase from Pseudomonas aeruginosa

1985 ◽  
Vol 230 (1) ◽  
pp. 227-237 ◽  
Author(s):  
N Foote ◽  
J Peterson ◽  
P M Gadsby ◽  
C Greenwood ◽  
A J Thomson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cytochrome C ◽  
High Spin ◽  
Spin State ◽  
High Potential ◽  
Reduced Form ◽  
Inactive Form ◽  
Temperature Spectra ◽  
State Changes ◽  
Haem Protein

Magnetic-c.d., e.p.r. and optical-absorption spectra are reported for the half-reduced form of Pseudomonas aeruginosa cytochrome c-551 peroxidase, a di-haem protein, and its fluoride derivative. Comparison of this enzyme species with oxidized peroxidase shows the occurrence of spin-state changes at both haem sites. The high-potential haem changes its state from partially high-spin to low-spin upon reduction. This is linked to a structural alteration at the ferric low-potential haem group, causing it to change from low-spin to high-spin. Low-temperature spectra demonstrate photolysis of an endogenous ligand of the high-potential haem. In addition, an inactive form of enzyme is examined in which the structural change at the ferric low-potential haem does not occur on reduction of the high-potential haem.

Control of proteoliposomal cytochrome c oxidase: the overall reaction

1990 ◽  
Vol 68 (9) ◽  
pp. 1128-1134 ◽  
Author(s):  
Peter Nicholls ◽  
Chris E. Cooper ◽  
John M. Wrigglesworth
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome C Oxidase ◽  
Respiration Rate ◽  
Respiratory Control ◽  
Ph Gradient ◽  
Activity Levels ◽  
Simulation Programme

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (ΔΨ) and pH gradient (ΔpH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse ΔΨ and increase ΔpH; the respiration rate decreases. High levels of valinomycin, however, decrease ΔpH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases ΔΨ and collapses ΔpH; the respiration rate increases. On a millivolt equivalent basis ΔpH is a more effective inhibitor of activity than is ΔΨ. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both ΔpH and ΔΨ components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing ΔpH but in a nonlinear way to ΔΨ ("non-ohmically"). High levels of both ΔΨ and ΔpH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both ΔpH and ΔΨ components of the proton-motive force, but is more sensitive to ΔpH than to ΔΨ at an equivalent ΔμH+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella™.Key words: cytochrome c, cytochrome oxidase, proteoliposomes, respiratory control, modelling, valinomycin, nigericin.

scholarly journals The reaction of Pseudomonas aeruginosa cytochrome c oxidase with sodium metabisulphite (Short Communication)

1974 ◽  
Vol 139 (1) ◽  
pp. 273-276 ◽  
Author(s):  
Stephen R. Parr ◽  
Michael T. Wilson ◽  
Colin Greenwood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Binding Sites ◽  
Short Communication ◽  
Sodium Metabisulphite

Spectrophotometric evidence is presented for the formation of a complex between metabisulphite and reduced Pseudomonas aeruginosa cytochrome c oxidase. The effects of metabisulphite on the recombination of CO with the reduced enzyme are discussed in terms of alternate binding sites for S2O52− and CO.

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