The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, reports on the amount of free NADH in mitochondria are sparse and contradictory. We first determined the emission spectrum of NADH bound to proteins using isothermal titration calorimetry combined with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins, and the amount is low in state 3 (substrate þ ADP present) and high in state 2 (only substrate present) and state 4 (substrate þ ATP). By contrast, the amount of free NADH is low but relatively constant, even increasing a little in state 3. Using modeling, we show that these results can be explained by a 2.5-to 3-fold weaker average binding of NADH to mitochondrial protein in state 3 compared with state 4. This indicates that there is a specific mechanism for free NADH homeostasis and that the concentration of free NADH in the mitochondrial matrix per se does not play a regulatory role in mitochondrial metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism.

An experimental investigation of the response of the multicomponent oxidative phosphorylation system to the environmental pollutant 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB) was performed by modular kinetic analysis in rat liver mitochondria oxidizing succinate (+ rotenone) and glutamate + malate. This approach facilitates the analysis of a complex process by dividing it into a small number of modules, each comprising multiple enzymatic steps, and allows evaluation of changes in the kinetics of individual blocks of the complex system induced by multisite effectors. Kinetic dependencies of the respiratory subsystem, the phosphorylation subsystem, and the proton permeability of the inner membrane on the membrane potential Delta Psi were determined in the control and in the presence of 20 microM 2,2',5,5'-TCB. The toxin inhibited the rate of respiration with both substrates to a similar extent (by 23-26%). We showed that 2,2',5,5'-TCB affected the all three modules of the oxidative phosphorylation system: it inhibited both the respiratory and the phosphorylation subsystems, and increased the membrane leak. As a result, the value of Delta Psi in State 3 of mitochondria oxidizing glutamate + malate remained the same or slightly increased with succinate, indicating that in the former case the respiratory subsystem was more sensitive to 2,2',5,5'-TCB. We explain this by the 2,2',5,5'-TCB-induced inhibition of Complex I. Moreover, 2,2',5,5'-TCB decreased the number of oligomycin-binding sites by 20%, caused a significant drop in the membrane potential generated by ATP hydrolysis, and inhibited activity of ATP hydrolysis in uncoupled mitochondria. Thus, we obtained evidence that at least one of the targets of 2,2',5,5'-TCB action within the phosphorylation module was ATP synthase.

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