ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa.

The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa. However, at physiological ionic strength (i.e. approximately 200 mM), activation by ATP was absent but inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP.     

Investigating the Mechanism of Electron Transfer to the Binuclear Center in Cu-Heme Oxidases

Novel experimental evidence is presented further supporting the hypothesis that, starting with resting oxidized cytochrome c oxidase, the internal electron transfer to the oxygen binding site is kinetically controlled. The reduction of the enzyme was followed spectroscopically and in the presence of NO or CO, used as trapping ligands for reduced cytochrome a₃; ruthenium hexamine was used as a spectroscopically silent electron donor. Consistent with the high combination rate constant for reduced cytochrome a₃, NO proved to be a very efficient trapping ligand, while CO did not. The results are discussed in view of two alternative (thermodynamic and kinetic) hypotheses of control of electron transfer to the binuclear (cyt.a₃-Cu_B) center. Fulfilling the prediction of the kinetic control hypothesis: i) the reduction of cytochrome a₃ and ligation are synchronous and proceed at the intrinsic rate of cytochrome a₃ reduction, ii) the measured rate of formation of the nitrosyl derivative is independent of the concentration of both the reductant and NO.     

Theoretical studies on control of oxidative phosphorylation in muscle mitochondria at different energy demands and oxygen concentrations

The mathematical dynamic model of oxidative phosphorylation in muscle mitochondria developed previously was used to calculate the flux control coefficients of particular steps of this process in isolated mitochondria at different amounts of hexokinase and oxygen concentrations. The pattern of control was completely different under different conditions. For normoxic concentration, the main controlling steps in state 4, state 3.5 and state 3 were proton leak, ATP usage (hexokinase) and complex III, respectively. The pattern of control in state 4 was not changed at hypoxic oxygen concentration, while in state 3.5 and state 3 much of the control was shifted from other steps to cytochrome oxidase. The implications of the theoretical results obtained for the regulation of oxidative phosphorylation in intact muscle are discussed.     

The dual role of oxygen in avocado fruit respiration: Kinetic analysis and computer modelling of diffusion-affected respiratory oxygen isotherms

Abstract. Oxygen plays a dual role in affecting the rate of respiration of avocado fruit ( Persea americana Mill, cv. Hass). The respiration rate v. oxygen concentration curve for steady state avocado fruit respiration is biphasic. The curve becomes monophasic, however, when measured under conditions of rapidly changing oxygen concentration in a closed circulating system. The results are interpreted as indicating that oxygen at relatively high concentrations modulates respiration independent of its interaction with the terminal oxidase—presumably cytochrome oxidase.
A computer model is presented which takes into account the effect of diffusion barriers on the kinetics of oxygen utilization as a function of concentration in avocado fruit. The model is used to make predictions concerning the apparent K _m of the terminal oxidase or oxidases in avocado fruit. It is concluded that the apparent K _m of the terminal oxidase of uninhibited avocado fruit is that of cytochrome oxidase, and that the alternative, cyanide-resistant oxidase of avocado fruit does not contribute appreciably to the uninhibited respiration of preclimacteric or climacteric avocado fruit.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Stimulation of the electron transport chain in mitochondria isolated from rats treated with mannoheptulose or glucagon

We investigated the kinetics of the mitochondrial respiratory chain, proton leak, and phosphorylating subsystems of liver mitochondria from mannoheptulose-treated and control rats. Mannoheptulose treatment raises glucagon and lowers insulin; it had no effect on the kinetics of the mitochondrial proton leak or phosphorylating subsystems, but the respiratory chain from succinate to oxygen was stimulated. Previous attempts to detect any stimulation of cytochrome c oxidase by glucagon are shown by flux control analysis to have used inappropriate assay conditions. To investigate the site of stimulation of the respiratory chain we measured the relationship between the thermodynamic driving force and respiration rate for the span succinate to coenzyme Q, the cytochrome bc1 complex and cytochrome c oxidase. Hormone treatment of rats altered the kinetics of electron transport from succinate to coenzyme Q in subsequently isolated mitochondria and activated succinate dehydrogenase. The kinetics of electron transport through the cytochrome bc1 complex were not affected. Effects on cytochrome c oxidase were small or nonexistent.     

The control of electron flux through cytochrome oxidase.

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.     

The polyphasic reduction of oxygen to water by purified cytochrome c oxidase

The time course of oxygen consumption by purified cytochrome oxidase has been studied in reactions where the fully reduced enzyme was rapidly mixed with molecular oxygen. Similar to intact mitochondria (Reynafarje & Davies, Am. J. Physiol. 258, 1990), the enzyme reduces oxygen to water in a kinetically and well defined polyphasic event. The initial rates of O2 consumption depended hyperbolically on O2 concentration, with a bimolecular rate constant of near 10(7) M-1 s-1. The Vmax of O2 uptake was, however, a complex function of the concentrations of both enzyme and cytochrome c. It is concluded that the reduction of oxygen to water takes place in a cyclic process in which the oxidase undergoes redox changes at rates depending on the relative concentration of the enzyme and its 3 substrates: O2, electrons and protons. No evidence was found for impairments in the intramolecular flow of electrons per se.     

Thermosensitive respiratory deficiency in yeast associated with specific effects on particulate cytochrome oxidase.

A mutant of Saccharomyces cerevisiae, unable to grow at the expense of non fermentable carbon sources at 37 degrees C, has been selected; at 25 degrees C the mutant strain behaves like the parental wild strain. Evaluations of respiration rates during aerobic growth at restrictive temperature on one hand, enzymatic and/or spectral evaluations of the individual components of the respiratory chain on the other hand show that the respiratory deficiency is specifically correlated with a reduced level of cytochrome oxidase. The decrease of enzyme activity is the direct consequence of a lowering of hemoprotein (a,a3) concentration. Temperature-activity relationship of cytochrome oxidase elaborated at the permissive temperature by the mutant strain is modified as far as the particulate enzyme is concerned, but no difference is observed after partial solubilization of the enzyme by non ionic surfactant. Genetic analysis shows that the mutant phenotype results from a nuclear gene mutation.     

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Explaining the enigmatic K(M) for oxygen in cytochrome c oxidase: a kinetic model

We present a mathematical model for the functioning of proton-pumping cytochrome c oxidase, consisting of cyclic conversions between 26 enzyme states. The model is based on the mechanism of oxygen reduction and linked proton translocation postulated by Wikstr?m and Verkhovsky (2007). It enables the calculation of the steady-state turnover rates and enzyme-state populations as functions of the cytochrome c reduction state, oxygen concentration, membrane potential, and pH on either side of the inner mitochondrial membrane. We use the model to explain the enigmatic decrease in oxygen affinity of the enzyme that has been observed in mitochondria when the proton-motive force is increased. The importance of the 26 transitions in the mechanism of cytochrome oxidase for the functional properties of cytochrome oxidase is compared through Metabolic Control Analysis. The control of the K(M) value is distributed mainly between the steps in the mechanism that involve electrogenic proton movements, with both positive and negative contributions. Positive contributions derive from the same steps that control enzyme turnover rate in the model. Limitations and possible further applications of the model are discussed.     

Change in Activity of Terminal Oxidases in The Roots of Rice Seedlings Under Oxygen-deficient Condition

Three-day old and seven-day old rice seedlings (Oryza sativa L. variety Yinfang) were treated under oxygen-deficient condition (by substituting the air with nitrogen in sealed glass bottles). Localization of cytochrome oxidase and polyphenol oxidase in roots were determined histochemically at fixed intervals after treatment (8, 24, 32, 48, 72 and 96 hours). The results obtained in this investigation may be briefly summarized as follows: 1. Polyphenol oxidase and cytochrome oxidase are detected in the seedlings from the second day of germination. 2. Maximum activity of polyphenol oxidase in the root tip appears on the fourth day of germination but it decreases progressively thereafter. In the vascular bundles of maturation zone, however, the enzyme activity is found to increase continuously with in- crease in age. If oxygen supply is limited or is withdrawn, a sharp decrease of enzyme activity occurs after 8 hours of treatment. It continues to decrease rapidly and almost disappears after about 48 hours. 3. The activity of cytochrome oxidase increases steadily up to the 10th day of germination when it reaches its plateau and is maintained at such a level thereafter. Under the condition of limited oxygen supply, the enzyme activity decreases but it still shows relative increases in activity as the plant grows. It appears likely that adaptive formation of this enzyme occurs during oxygen deficiency. On the basis of the observed results, one may suggest that the adaptation of cytochrome oxidase to limited oxygen supply probably plays an important role in the tolerance of rice to submergence under water. 4. The results show that these two types of terminal oxidases are more sensitive to oxygen on the third day than on the seventh day of germination.     

Parallel activation in the ATP supply-demand system lessens the impact of inborn enzyme deficiencies, inhibitors, poisons or substrate shortage on oxidative phosphorylation in vivo

A potential kinetic impact of parallel activation of different steps during an increased energy demand on the effect of inborn enzyme deficiencies, physiological inhibitors, external poisons and substrate shortage on oxidative phosphorylation was studied in the theoretical way. Numerical simulations were performed with the aid of the previously developed computer model of oxidative phosphorylation. It was demonstrated that the parallel activation mechanism diminishes significantly changes in fluxes and metabolite concentrations occurring at a given degree of inactivation of the system by one of the above-mentioned factors. It was also shown that parallel activation decreases greatly the threshold value of the relative activity of oxidative phosphorylation, below which the oxygen consumption flux and ATP turnover flux become significantly affected. Finally, computer simulations predicted that parallel activation leads to a considerable increase in the apparent affinity of oxidative phosphorylation to oxygen, which delays the effect of inhibitors and poisons competing with oxygen for the active centre of cytochrome oxidase. It is concluded that one of possible functions of parallel direct activation of different steps of oxidative phosphorylation is to increase the resistance of the system to a decrease in the concentration/activity of different oxidative phosphorylation complexes.     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen metabolism and a potential role for cytochrome c oxidase in the Warburg effect

By manipulating the physical properties of oxygen, cells are able to harvest the large thermodynamic potential of oxidation to provide a substantial fraction of the energy necessary for cellular processes. The enzyme largely responsible for this oxygen manipulation is cytochrome c oxidase, which resides at the inner mitochondrial membrane. For unknown reasons, cancer cells do not maximally utilize this process, but instead rely more on an anaerobic-like metabolism demonstrating the so-called Warburg effect. As the enzyme at the crossroads of oxidative metabolism, cytochrome c oxidase might be expected to play a role in this so-called Warburg effect. Through protein assay methods and metabolic studies with radiolabeled glucose, alterations associated with cancer and cytochrome c oxidase subunit levels are explored. The implications of these findings for cancer research are discussed briefly.     

Respiratory Development in Saccharomyces cerevisiae Grown at Controlled Oxygen Tension

Saccharomyces cerevisiae was grown in batch culture over a wide range of oxygen concentrations, varying from the anaerobic condition to a maximal dissolved oxygen concentration of 3.5 muM. The development of cells was assayed by measuring amounts of the aerobic cytochromes aa(3), b, c, and c(1), the cellular content of unsaturated fatty acids and ergosterol, and the activity of respiratory enzyme complexes. The half-maximal levels of membrane-bound cytochromes aa(3), b, and c(1), were reached in cells grown in O(2) concentrations around 0.1 muM; this was similar to the oxygen concentration required for half-maximal levels of unsaturated fatty acid and sterol. However, the synthesis of ubiquinone and cytochrome c and the increase in fumarase activity were essentially linear functions of the dissolved oxygen concentration up to 3.5 muM oxygen. The synthesis of the succinate dehydrogenase, succinate cytochrome c reductase, and cytochrome c oxidase complexes showed different responses to changes in O(2) concentration in the growth medium. Cyanide-insensitive respiration and P(450) cytochrome content were maximal at 0.25 muM oxygen and declined in both more anaerobic and aerobic conditions. Cytochrome c peroxidase and catalase activities in cell-free homogenates were high in all but the most strictly anaerobic cells.     

Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism

In metabolism research, thermodynamics is usually used to determine the directionality of a reaction or the feasibility of a pathway. However, the relationship between thermodynamic potentials and fluxes is not limited to questions of directionality: thermodynamics also affects the kinetics of reactions through the flux-force relationship, which states that the logarithm of the ratio between the forward and reverse fluxes is directly proportional to the change in Gibbs energy due to a reaction (Δ_rG′). Accordingly, if an enzyme catalyzes a reaction with a Δ_rG′ of -5.7 kJ/mol then the forward flux will be roughly ten times the reverse flux. As Δ_rG′ approaches equilibrium (Δ_rG′ = 0 kJ/mol), exponentially more enzyme counterproductively catalyzes the reverse reaction, reducing the net rate at which the reaction proceeds. Thus, the enzyme level required to achieve a given flux increases dramatically near equilibrium. Here, we develop a framework for quantifying the degree to which pathways suffer these thermodynamic limitations on flux. For each pathway, we calculate a single thermodynamically-derived metric (the Max-min Driving Force, MDF), which enables objective ranking of pathways by the degree to which their flux is constrained by low thermodynamic driving force. Our framework accounts for the effect of pH, ionic strength and metabolite concentration ranges and allows us to quantify how alterations to the pathway structure affect the pathway''s thermodynamics. Applying this methodology to pathways of central metabolism sheds light on some of their features, including metabolic bypasses (e.g., fermentation pathways bypassing substrate-level phosphorylation), substrate channeling (e.g., of oxaloacetate from malate dehydrogenase to citrate synthase), and use of alternative cofactors (e.g., quinone as an electron acceptor instead of NAD). The methods presented here place another arrow in metabolic engineers'' quiver, providing a simple means of evaluating the thermodynamic and kinetic quality of different pathway chemistries that produce the same molecules.     

Oxygen and proton pathways in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump, which couples the reduction of oxygen to water to the translocation of protons across the membrane. The recently solved x-ray structures of cytochrome c oxidase permit molecular dynamics simulations of the underlying transport processes. To eventually establish the proton pump mechanism, we investigate the transport of the substrates, oxygen and protons, through the enzyme. Molecular dynamics simulations of oxygen diffusion through the protein reveal a well-defined pathway to the oxygen-binding site starting at a hydrophobic cavity near the membrane-exposed surface of subunit I, close to the interface to subunit III. A large number of water sites are predicted within the protein, which could play an essential role for the transfer of protons in cytochrome c oxidase. The water molecules form two channels along which protons can enter from the cytoplasmic (matrix) side of the protein and reach the binuclear center. A possible pumping mechanism is proposed that involves a shuttling motion of a glutamic acid side chain, which could then transfer a proton to a propionate group of heme α₃. Proteins 30:100–107, 1998. © 1998 Wiley-Liss, Inc.     

The effect of nitrite on cytochrome oxidase

Nitrite inhibits the oxygen uptake by the system ferrocytochrome c-cytochrome oxidase with Ki = 1.5 mM. In the absence of ferrocytochrome c the oxygen uptake by cytochrome oxidase in the presence of nitrite was observed indicating that the enzyme has some nitrite oxidase activity. Nitrite induces changes in optical difference spectra of cytochrome oxidase and, in particular, the formation of the transient band at 607 nm. The reciprocal relation was observed between the intensity of this band and the rate of the oxygen uptake by cytochrome oxidase. This means that the form of the enzyme with this band does not involved in the nitrite oxidase activity. It is suggested that the nitrite oxidase activity relates to the oxygen binding site rather than the cytochrome c binding site of the enzyme.