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

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.

Download Full-text

The control of electron flux through cytochrome oxidase

Biochemical Journal ◽

10.1042/bj2430499 ◽

1987 ◽

Vol 243 (2) ◽

pp. 499-505 ◽

Cited By ~ 36

Author(s):

M P Murphy ◽

M D Brand

Keyword(s):

Cytochrome C ◽

Cytochrome Oxidase ◽

Linear Function ◽

Redox Reaction ◽

Electron Flux ◽

Limited Range ◽

Thermodynamic Force ◽

Percentage Reduction ◽

Wide Range

1. The electron flux through cytochrome oxidase is a linear function of the net thermodynamic force across the complex over a limited range of conditions. 2. Over a wide range of conditions the electron flux is a complicated function of the percentage reduction of the cytochrome c pool and of delta psi (at low values of delta pH). 3. We have estimated the elasticities of electron flux through cytochrome oxidase to delta Eh of the redox reaction catalysed by cytochrome oxidase (or to cyt c2+/cyt c3+) and to delta psi. The elasticities varied depending on the values of delta psi and of the percentage reduction of the cytochrome c pool. 4. At intermediate rates (which may correspond to those in vivo) the electron flux through cytochrome oxidase is controlled to about the same extent by delta psi and by delta Eh.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2300797 ◽

1985 ◽

Vol 230 (3) ◽

pp. 797-805 ◽

Cited By ~ 25

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.

Download Full-text

Control of proteoliposomal cytochrome c oxidase: the partial reactions

Biochemistry and Cell Biology ◽

10.1139/o90-169 ◽

1990 ◽

Vol 68 (9) ◽

pp. 1135-1141 ◽

Cited By ~ 4

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.

Download Full-text

The oxidized forms of cytochrome oxidase

Canadian Journal of Biochemistry ◽

10.1139/o68-207 ◽

1968 ◽

Vol 46 (11) ◽

pp. 1371-1379 ◽

Cited By ~ 24

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.

Download Full-text

Control of proteoliposomal cytochrome c oxidase: the overall reaction

Biochemistry and Cell Biology ◽

10.1139/o90-168 ◽

1990 ◽

Vol 68 (9) ◽

pp. 1128-1134 ◽

Cited By ~ 7

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.

Download Full-text