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Impaired mitochondrial function contributes to copper- and cadmium-induced cellular dysfunction. In this study, we used modular kinetic analysis and metabolic control analysis to assess how Cd

Modular kinetic analysis was used to determine the sites in plant mitochondria where charge-screening stimulates the rate of electron transfer from external NAD(P)H to oxygen. In mitochondria isolated from potato (Solanum tuberosum L.) tuber callus, stimulation of the rate of oxygen uptake was accompanied by a decrease in the steady-state reduction level of coenzyme Q, and by a small decrease in the steady-state reduction level of cytochrome c. Modular kinetic analysis around coenzyme Q revealed that stimulation of the rate was due to stimulation of quinol oxidation via the cytochrome pathway (cytochrome bc

In this work we demonstrate how the reduction state of the Q-pool determines the distribution of electron flow over the two quinol-oxidising branches in Paracoccus denitrificans: one to quinol oxidase, the other via the cytochrome bc1 complex to the cytochrome c oxidases. The dependence of the electron-flow rate to oxygen on the fraction of quinol in the Q-pool was determined in membrane fractions and in intact cells of the wild-type strain, a bc1-negative mutant and a quinol oxidase-negative mutant. Membrane fractions of the bc1-negative mutant consumed oxygen at significant rates only at much higher extents of Q reduction than did the wild-type strain or the quinol oxidase-negative mutant. In the membrane fractions, dependence on the Q redox state was exceptionally strong corresponding to elasticity coefficients close to 2 or higher. In intact cells, the dependence was weaker. In uncoupled cells the dependence of the oxygen-consumption rates on the fractions of quinol in the Q-pool in the wild-type strain and in the two mutants came closer to that found for the membrane fractions. We also determined the dependence for membrane fractions of the wild-type in the absence and presence of antimycin A, an inhibitor of the bc1 complex. The dependence in the presence of antimycin A resembled that of the bc1-negative mutant. These results indicate that electron-flow distribution between the two quinol-oxidising branches in P. denitrificans is not only determined by regulated gene expression but also, and to a larger extent, by the reduction state of the Q-pool.

Much of enzyme kinetics builds on simplifications enabled by the quasi-steady-state approximation and is highly useful when the concentration of the enzyme is much lower than that of its substrate. However, in vivo, this condition is often violated. In the present study, we show that, under conditions of realistic yet high enzyme concentrations, the quasi-steady-state approximation may readily be off by more than a factor of four when predicting concentrations. We then present a novel extension of the quasi-steady-state approximation based on the zero-derivative principle, which requires considerably less theoretical work than did previous such extensions. We show that the first-order zero-derivative principle, already describes much more accurately the true enzyme dynamics at enzyme concentrations close to the concentration of their substrates. This should be particularly relevant for enzyme kinetics where the substrate is an enzyme, such as in phosphorelay and mitogen-activated protein kinase pathways. We illustrate this for the important example of the phosphotransferase system involved in glucose uptake, metabolism and signaling. We find that this system, with a potential complexity of nine dimensions, can be understood accurately using the first-order zero-derivative principle in terms of the behavior of a single variable with all other concentrations constrained to follow that behavior. © 2009 FEBS.

We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24 ± 6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N

Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Δψ) and cytosolic ATP concentrations. We here show that 5 μm palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (QH

Modular kinetic analysis reveals that the environmental pollutant 2,2′,5,5′tetrachlorobiphenyl (2,2′,5,5′-TCB) affects a large number of steps in oxidative phosphorylation in rat liver mitochondria. 2,2′,5,5′-TCB increases membrane permeability to ions, and inhibits NADH dehydrogenase, cytochrome bcl, cytochrome oxidase (all in the respiratory chain) and ATP-synthase (in the phosphorylation subsystem). Surprisingly, flux control distribution does not change. A kinetic model for oxidative phosphorylation was used to simulate these findings, and it was found that combined large changes in the processes indicated indeed left the flux control largely unchanged. In addition, computational analysis with the model indicated that the adenine nucleotide translocator might be inhibited by 2,2′,5,5′-TCB.

Regulation of succinate dehydrogenase was investigated using tightly coupled potato tuber mitochondria in a novel fashion by simultaneously measuring the oxygen uptake rate and the ubiquinone (Q) reduction level. We found that the activation level of the enzyme is unambiguously reflected by the kinetic dependence of the succinate oxidation rate upon the Q-redox poise. Kinetic results indicated that succinate dehydrogenase is activated by both ATP (K