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.

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 Titration and steady-state behaviour of the 830 nm chromophore in cytochrome c oxidase

1982 ◽  
Vol 203 (3) ◽  
pp. 541-549 ◽  
Author(s):  
P Nicholls ◽  
G A Chanady
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Lag Phase ◽  
State Reduction ◽  
Cytochrome Aa3 ◽  
System A ◽  
Reducing Equivalent ◽  
State Behaviour ◽  
Copper Excess

Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN'N'-tetramethyl-p-phenylenediamine initially introduces two reducing equivalents per mol of cytochrome aa3. The first equivalent reduces the cytochrome a haem iron; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (e.p.r.-detectable copper) but is probably required for reduction of the e.p.r.-undetectable copper. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with ab increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. It is concluded that the e.p.r.-undetectable copper (CuD) is reduced early in the reaction sequence, whereas the detectable copper (CUD) is reduced late. The latter species is probably that responsible for reduction of the cytochrome a3 haem. The magnetic association between undetectable copper and the a3 haem may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

1992 ◽  
Vol 70 (5) ◽  
pp. 301-308 ◽  
Author(s):  
M. Crinson ◽  
P. Nicholls
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Reduction Rate ◽  
Artificial Substrates ◽  
Intramolecular Electron Transfer ◽  
State Reduction ◽  
Hydrogen Donors ◽  
Slow Turnover

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT – cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (≈70% of the total with resting and ≈85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.Key words: cytochrome c oxidase, cytochrome a, respiration, cyanide, stopped flow.

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.

Effects of triorganotin-mediated anion–hydroxide exchange upon reconstituted cytochrome c oxidase proteoliposomes

1986 ◽  
Vol 64 (7) ◽  
pp. 647-655 ◽  
Author(s):  
A. P. Singh ◽  
P. Nicholls
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiration Rate ◽  
Ph Gradient ◽  
Dye Uptake ◽  
Tin Chloride ◽  
Carbocyanine Dye ◽  
Potential Formation

Proteoliposomes containing cytochrome c oxidase and an internally trapped fluorescent pH probe (pyranine) were used to monitor respiration-dependent internal alkalinization and membrane potential formation. A maximum steady-state pH gradient of about 0.4 pH unit (vesicle interior alkaline) was obtained during active respiration in presence of reducing substrates and cytochrome c. This pH gradient was abolished by the triorganotin compounds tripropyl-, tributyl-, and triphenyl-tin chloride. At the same time, the membrane potential, measured by carbocyanine dye uptake, was slightly increased in value. Valinomycin, which abolishes the membrane potential, restores the value of ΔpH at low trialkyltin concentrations. The organotin compounds acted as electroneutral ionophores which exchanged intravesicular OH− ions with external SCN−, I−, and Cl− ions, but not [Formula: see text] or [Formula: see text] ions. Abolition of ΔpH is accompanied by an increase in respiration rate, but full resiratory stimulation only occurs when both Δψ and ΔpH are abolished by addition of both triorganotin and valinomycin. The triorganotin–valinomycin combination leads to active KC1 accumulation by the respiring proteoliposome, and it is necessary to postulate an electrically neutral KC1 efflux process to explain the continued steady respiration of the proteoliposomes in the presence of this ionophore combination.

Comparative studies on succinate and terminal oxidase activity in microbial and mammalian electron-transport systems

1969 ◽  
Vol 15 (7) ◽  
pp. 797-807 ◽  
Author(s):  
Peter Jurtshuk ◽  
Ann K. May ◽  
Leodocia M. Pope ◽  
Patricia R. Aston
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Comparative Studies ◽  
Azotobacter Vinelandii ◽  
Transport Systems ◽  
Terminal Oxidase ◽  
Succinate Oxidation ◽  
State Reduction ◽  
Hydroxy Quinoline ◽  
Terminal Oxidation

A comparative study was undertaken to examine the succinate and terminal oxidase activities of the electron-transport systems of Azotobacter vinelandii and mammalian mitochondria. For succinate oxidation, both systems exhibited similar relative specificities for the electron acceptors phenazine methosulfate, O2, methylene blue, K3Fe(CN)6, nitrotetrazolium blue, 2,6-dichlorophenolindophenol (DCIP), and cytochrome c. They differed in that DCIP and cytochrome c were less active in the Azotobacter electron-transport system (R3 fraction) than in the bovine mitochondrial system. Comparative studies with known inhibitors of mammalian mitochondrial electron-transport demonstrated that the succinoxidase activity of the Azotobacter R3 fraction was, at least, 2000 times less sensitive to antimycin A, 700 times less sensitive to thenoyl-trifluoroacetone, and 30 times less sensitive to 2-n-heptyl-4-hydroxy-quinoline-N-oxide. Both systems were equally sensitive to KCN, p-chloromercuribenzoic acid, and chlorpromazine.The ability of the two systems to use tetramethyl-p-phenylenediamine (TMPD) and its derivatives as electron donors, for terminal oxidation, was also similar. Studies on steady state reduction revealed that in the Azotobacter R3 fraction, the cytochromes (a2, a1, b1, c4 + c5) and flavoprotein components were reduced substantially by succinate as well as by TMPD in the presence of ascorbate. Ultrastructure analyses of the Azotobacter R3 electron-transport fraction revealed the vesicular membranous components identified as oxidosomes according to the terminology used by DeLey and contained spherical headpiece units of 80 Å in diameter which appeared to be morphologically identical with the tripartite units or the elementary particles described by Green and associates, viz., Kopaczyk et al., and by Fernandez-Moran et al.

Response of Heme Symmetry to the Redox State of Bovine Cytochrome c Oxidase

Biochemistry ◽  
2018 ◽  
Vol 57 (28) ◽  
pp. 4105-4113
Author(s):  
Katarina Kopcova ◽  
Ludmila Blascakova ◽  
Tibor Kozar ◽  
Daniel Jancura ◽  
Marian Fabian
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Redox State
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