Kinetics Of Cytochrome Oxidase Research Articles

Measuring and interpreting respiratory critical oxygen pressures in roots.

Respiratory critical oxygen pressures (COPR) determined from O(2)-depletion rates in media bathing intact or excised roots are unreliable indicators of respiratory O(2)-dependency in O(2)-free media and wetlands. A mathematical model was used to help illustrate this, and more relevant polarographic methods for determining COPR in roots of intact plants are discussed. Cortical [O(2)] near the root apex was monitored indirectly (pea seedlings) from radial oxygen losses (ROL) using sleeving Pt electrodes, or directly (maize) using microelectrodes; [O(2)] in the root was controlled by manipulating [O(2)] around the shoots. Mathematical modelling of radial diffusive and respiratory properties of roots used Michaelis-Menten enzyme kinetics. Respiration declined only when the O(2) partial pressure (OPP) in the cortex of root tips fell below 0.5-4.5 kPa, values consistent with depressed respiration near the centre of the stele as confirmed by microelectrode measurements and mathematical modelling. Modelling predictions suggested that the OPP of a significant core at the centre of roots could be below the usual detection limits of O(2)-microelectrodes but still support some aerobic respiration. In O(2)-free media, as in wetlands, the COPR for roots is likely to be quite low, dependent upon the respiratory demands, dimensions and diffusion characteristics of the stele/stelar meristem and the enzyme kinetics of cytochrome oxidase. Roots of non-wetland plants may not differ greatly in their COPRs from those of wetland species. There is a possibility that trace amounts of O(2) may still be present in stelar 'anaerobic' cores where fermentation is induced at low cortical OPPs.

Myocardial cytochrome oxidase activity is decreased following carbon monoxide exposure

Carbon monoxide (CO) inhalation often leads to cardiac dysfunction, dysrhythmias, ischemia, infarction, and death. However, the underlying mechanism of CO toxicity is poorly understood. We hypothesize that inhaled CO interrupts myocardial oxidative phosphorylation by decreasing the activity of myocardial cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain. Male C57Bl6 mice were exposed to either 1000 ppm (0.1%) CO or air for 3 h. Cardiac ventricles were harvested and mitochondria were isolated. CcOX kinetics and heme aa 3 content were measured. V max, K m, and turnover number were determined. Levels of CcOX subunit I message and protein were evaluated. Carboxyhemoglobin (COHb) levels were measured and tissue hypoxia was assessed with immunohistochemistry for pimonidazole hydrochloride. CO significantly decreased myocardial CcOX activity and V max without altering K m. Heme aa 3 content and CcOX I protein levels significantly decreased following CO exposure while enzyme turnover number and CcOX I mRNA levels remained unchanged. CO exposure increased COHb levels without evidence of tissue hypoxia as compared to sham and hypoxic controls. Decreased CcOX activity following CO inhalation was likely due to decreased heme aa 3 and CcOX subunit I content. Importantly, myocardial CcOX impairment could underlie CO induced cardiac dysfunction.

Simulation of oxidative phosphorylation in hepatocytes

The dynamic mathematical model of oxidative phosphorylation proposed previously was modified, developed and further tested. The description of cytochrome oxidase kinetics was changed to involve dependence on Δp. Simple, phenomenological descriptions of the kinetics of substrate dehydrogenation and ATP usage, able to reflect experimental data correctly, were found. The kinetic response of the oxidation subsystem (substrate dehydrogenation, respiratory chain), phosphorylation subsystem (ATP synthase, ATP ADP carrier, phosphate carrier, ATP usage) and proton leak to the changes of Δp in isolated hepatocytes incubated with different respiratory substrates was simulated. The simulations revealed a good agreement with the experimental results. Simple, intuitive assumptions were able, when introduced into the model, to explain differences in the properties of the oxidative phosphorylation system working with different respiratory substrates. It was proposed, therefore, that our explicit understanding of the oxidative phosphorylation system was good enough to explain many properties of this system correctly, at least in the range of physiological conditions tested.

Nature of respiratory stimulation in hyperthyroidism: the redox behaviour of cytochrome c

Hyperthyroid mitochondria show an increased K m and V max in the high affinity phase of cytochrome oxidase kinetics. During inhibitor titrations, cytochrome c shows a different redox behaviour in hyperthyroid with respect to protonophore-treated euthyroid mitochondria. The observations are discussed in terms of a different regulation of electron input and output into the respiratory chain during slip and leak types of uncoupling. In hyperthyroid mitochondria during inhibitor titrations, the pattern of the relationship between uncoupler-induced extra-respiration and membrane potential is highly non-linear. The complex nature of the respiratory stimulation in hyperthyroid mitochondria is discussed.

Control of cytochrome oxidase activity. A transient spectroscopy study

The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.

Modulation of cytochrome oxidase kinetics by indirect antibody action

Polyclonal antibodies raised against isolated subunit V from beef heart cytochrome oxidase or against the intact enzyme increase its apparent affinity for the substrate cytochrome c at the high-affinity site while diminishing the turnover at that site. At the low-affinity site the major action of both types of antibody is to reduce the apparent affinity for cytochrome c. At high ionic strengths the kinetic effect of anti-subunit V is very small although it still binds to the enzyme. The results are interpreted in terms of a model for the enzyme in which antibodies can modulate cytochrome oxidase kinetics by affecting the binding of cytochrome c, even if the antibody-binding site is on a subunit not directly involved in substrate binding.

The kinetic mechanism(s) of cytochrome oxidase. Techniques for their analysis and criteria for their validation.

The steady-state kinetics of cytochrome oxidase exhibit two characteristics that impose severe constraints on any proposed mechanism. The first is the exponential consumption of ferrocytochrome c and the second is the nonhyperbolic dependence of reaction velocity upon the concentration of cytochrome c. Because the reaction mechanism contains at least five, and possibly six, substrates, realistic mechanisms can be very complex and not suitable for analysis by conventional means. We have developed procedures for rapidly establishing whether a postulated mechanism will exhibit the necessary behavior and for calculating the steady-state activity that will result for any mechanism, given values for the individual rate constants and reactant concentrations. The procedures have been used with mechanisms containing up to 40 enzyme species.

Effect of naphthalene on cytochrome oxidase activity.

Previous reports have demonstrated that naphthalene inhibits oxygen consumption in Daphnia magna tissue culture cells, and intact mitochondria and submitochondrial particles. These studies were extended to algal mitochondrial respiration as well as photosynthetic activity. The authors were able to demonstrate the specific site of apparent respiratory inhibition to be coenzyme Q (ubiquinone, UQ) and later to demonstrate the molecular basis of this inhibition at ubiquinone. The authors previously could not demonstrate an effect of naphthalene on cytochrome oxidase activity. However, the observation that naphthalene can stimulate respiration in algae prompted the reinvestigation of the effect of naphthalene on the kinetics of cytochrome oxidase. Cytochrome oxidase is a multi-subunit membranous protein responsible for the oxidation of cytochrome c and the reduction of molecular oxygen to water. Because of the complicated nature and mechanism of this enzyme, the potential exists for multiple and possibly opposite effects of naphthalene on its function.

Electron-transport-driven proton pumps display nonhyperbolic kinetics: Simulation of the steady-state kinetics of cytochrome c oxidase

A reaction cycle for electron-transportdriven proton pumps is proposed. It includes two distinct conformational states of the pump protein in which the primary electron acceptor has different reduction potentials. This has as an unavoidable consequence that the steady-state rate equation for the catalytic reaction driving the pump is nonhyperbolic. The model can be used to simulate experimental results for the kinetics of cytochrome oxidase (EC 1.9.3.1) in a wide range of experimental conditions (ionic strength, pH, temperature). It is thus not necessary to invoke more than one binding site for cytochrome c to account for the biphasic response of the oxidase activity to the concentration of this substrate.

Oxygen "pulsed" cytochrome c oxidase: functional properties and catalytic relevance.

The kinetics of the reaction of cytochrome c with solubilized mammalian cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) has been studied by a stopped-flow technique under two different experimental situations: (i) the completely oxidized enzyme (resting oxidase as obtained from the preparation) was mixed with reduced cytochrome c, and (ii) the completely reduced enzyme in the presence of reduced cytochrome c was exposed to a "pulse" of O2 (pulsed oxidase). Both sets of experiments were performed with either "limiting" or "excess" O2 (relative to oxidase), in the presence or absence of CO. Both the pre-steady-state events and the steady-state kinetics of cytochrome oxidase are found to be different in the two cases. This shows that the product of the reaction of fully reduced oxidase with O2 (pulsed oxidase) is functionally different from the oxidase as prepared (resting oxidase). These differences are interpreted with the assumption of a different rate of intramolecular electron transfer in the pulsed and resting oxidases. Implications of these experimental findings are discussed in the general framework of a tentative model for the catalytic cycle of the oxidase.

The solid state physical theory of cytochrome oxidase kinetics. Inhibition of second order rate constant, and second to first order kinetic shift with increasing oxygen, predicted from electron injection and trapping

Conduction of electrons through the solid protein cytochrome oxidase particle in accord with Ohm's law, driven by the difference in electrode potentials of two substrates which exchange electrons with the two sides of the enzyme particle, was previously shown to explain the inhibitory effect of cytochromec on the first order rate constant, and to predict the low semiconduction activation energy of dried cytochrome oxidase. If the solid conduction path in the cytochrome oxidase particle shows electron injection from sites of electron exchange with substrate, and shows trapping of conduction electrons by reversible O2 complexes, then one may also predict that the first order kinetics observed as high O2 concentrations will change to second order kinetics at lower O2 concentrations, as observed by Gibson and Wharton. One may also predict quantitatively the inhibitory effect of increasing O2 concentrations on the second order rate constant as observed by Gibson and Wharton. The same concept of electron trapping by O2 complexes provides a possible reason for the unusually low semiconduction activation energy of cytochrome oxidase.

Flow flash kinetics of the cytochrome a3-oxygen reaction in coupled and uncoupled mitochondria using the liquid dye laser

The duality of crossover responses of cytochromes a 3 and c indicate energy-coupling sites between c and the oxidase, and between the oxidase and oxygen. Thermodynamic data indicate that the a 3 component specifically responds to ATP with a decrease of redox potential of approximately 100 mV. A kinetic analysis of cytochrome oxidase in intact mitochondrial membranes seems necessary to identify the kinetic repercussions of the steady state and thermodynamic observations. A special form of the dualwavelength spectrophotometric technique termed “cycle selection” has been devised to permit the recording of absorbancy changes from 150 μsec onward. The new technique is compatible with the existing high-precision 60-Hz light modulation of the instrument and allows its application to flow-flash photolysis methods which employ the 0.4-μsec pulses of 585-nm light from the recently developed liquid dye laser. The data indicate precisely the slope and the halftime of the cytochrome oxidase kinetics in the time range of a few tenths of a millisecond to 8 msec. The oxidation of cytochrome a 3 under experimental conditions employed is substantially complete at 3 msec, the corresponding second-order velocity constant is 3.3 × 10 7 m −1 sec −1 at 24 ° in good agreement with the previous determinations. Addition of ATP causes nearly complete oxidation of cytochrome a and partial oxidation of a 3 in the anaerobic system due to reversed electron flow. In addition, it decreases the slope of the a 3 kinetics by a factor of 3. Since the previous data have shown slowing of cytochrome c oxidation by ATP (time range of 8–25 msec) this observation affords an explanation for the existence of crossover points between cytochrome a 3 and oxygen and cytochromes c and a. Thus, control of electron flow operates on both the oxidizing and reducing reactions of the cytochrome oxidase.