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First published online February 3, 2006; 10.1105/tpc.105.039354 The Plant Cell 18:688-698 (2006) © 2006 American Society of Plant Biologists The Free NADH Concentration Is Kept Constant in Plant Mitochondria under Different Metabolic Conditions
a Optics and Plasma Research Department, Risø National Laboratory, DK-4000 Roskilde, Denmark 1 To whom correspondence should be addressed. E-mail imm{at}kvl.dk; fax 45-3528-3460.
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 + ADP present) and high in state 2 (only substrate present) and state 4 (substrate + 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.
NAD and NADP are two of the most used coenzymes in cellular metabolism. More than 500 known enzymes use NAD(P) to catalyze reduction-oxidation reactions reversibly (Enzyme Nomenclature; http://www.chem.qmul.ac.uk/iubmb/enzyme). Some of these are among the most abundant and well-studied enzymes participating in energy metabolism (glycolysis, the Krebs cycle, the Calvin cycle), biosynthesis, degradation, defense against oxidative damage, etc. NAD(P) is found in mitochondria, chloroplasts, peroxisomes, the cytosol, and other cellular compartments, typically at a total concentration of one to several millimolar.
The reduced and oxidized forms of the two coenzymes have distinct spectroscopic characteristics—NAD(P)H is fluorescent, while its oxidized form is not. Furthermore, the fluorescence properties of bound and free NAD(P)H also differ. Free NADH has an emission peak at 460 nm and a lifetime of 0.4 ns (Scott et al., 1970
Despite the importance of NAD(P) in mitochondrial metabolism, little is known about the amount of free NAD(P)H, the parameter important for the interaction of the coenzyme with enzymes. Wakita et al. (1995)
Mitochondrial Respiration with Malate (+Glu) as Substrate Isolated potato (Solanum tuberosum cv Bintje) tuber mitochondria were incubated in a respiratory medium, and substrate and ADP were added consecutively. In the following work, we will use the nomenclature for respiratory states of isolated mitochondria introduced by Chance and Williams (see Nicholls and Ferguson, 2002  ): State 1, mitochondria alone; state 2, only substrate added; state 3, substrate and a limited amount of ADP added; state 4, substrate and ATP added (or all ADP converted into ATP).
The oxygen consumption traces show the expected pattern (Figure 1A): There was little or no oxygen consumption until substrate was added. The mitochondria started respiring after the addition of substrate (Glu + malate; state 2), but the rate of oxidation was greatly enhanced when ADP was added (state 3). When the ADP was exhausted, the rate of oxidation became approximately fourfold slower (state 4), indicating that electron transport and ATP synthesis were well coupled in these mitochondria. After further addition of ADP, the pattern was repeated. In the example shown in Figure 1A, both states 3 and 4 were linear. However, state 4 sometimes showed two phases, slowest in the first half and faster after 2 to 3 min. This is well known for plant mitochondria oxidizing malate, and it is due to accumulation of oxaloacetate in state 3 and its removal in state 4 (Møller and Palmer, 1982
NADH Fluorescence during Malate Oxidation The amount of free and bound NADH was monitored by exciting the mitochondria with nanosecond laser pulses at 355 nm and monitoring the fluorescence through the wall of the oxygen electrode vessel (see Methods). Emission spectra were collected every 4 s, and the data at 420, 460, and 500 nm are shown in Figure 1B as a function of time. Note that uncorrected spectra and uncorrected wavelengths are given here and elsewhere in Results. As demonstrated in Methods, the corrected peaks are blue-shifted by  30 nm.
Upon addition of Glu and malate, the level of fluorescence intensity increased sharply at all wavelengths. The addition of ADP caused fluorescence to decrease throughout the spectrum but more so in the blue region. At the time of transition from state 3 to state 4 at 200 s, the fluorescence started increasing and reached the state 2 levels after a couple of minutes. This fluorescence response is perfectly consistent with the data obtained at fixed wavelengths in earlier investigations (Neuburger et al., 1984
A puzzling feature of our data recurring in most of the samples was the initial fluorescence shown by mitochondria prior to the addition of substrates. The shape of these emission spectra was characteristic of the free NADH. However, extraction and quantification of NAD+ and NADH by an analytical method showed clearly the absence of NADH in state 1, a high amount in states 2 and 4, and a low amount in state 3 (Table 1). Excitation fluorescence spectra of the mitochondria (data not shown) in state 1 also confirmed that they contained no NADH. This initial fluorescence is not associated with NADPH because no NADPH is detectable in isolated potato tuber mitochondria under any metabolic conditions (Agius et al., 2001
Upon binding to a protein, the NADH emission maximum shifts to shorter wavelengths, while the fluorescence intensity increases fourfould to fivefold (see Methods; Duysens and Kronenberg, 1957 ; Langan, 1960; Avi-Dor et al., 1962; Wakita et al., 1995). The fluorescence spectra of potato tuber mitochondria, uncorrected for the instrument intensity response function, are shown in Figure 1C. After correction for instrument response, the emission maximum for free NADH is at 460 to 470 nm, perfectly consistent with the literature (see Methods).
Upon addition of Glu + malate (state 2), the fluorescence intensity increased twofold (Figures 1B and 1C), and the emission peak blue-shifted
Determination of the Fluorescence Spectrum of Bound NADH in Respiring Mitochondria
Changes in Bound and Free NADH in Response to Respiratory State
The transition in respiration between state 3 and state 4, as monitored by oxygen consumption, was rapid. Upon exhaustion of ADP, it took <20 s for the rate of oxygen consumption to reach a new steady state. Likewise, the faster linear state 3 rate was attained very quickly after ADP addition (Figure 1A). Clearly, the closure of the proton-conducting pore of the ATP synthase and the subsequent rise in the size of the membrane potential had an immediate effect upon the rate of electron transfer to oxygen. By contrast, the amount of NADH took 2 min to reach its new steady state after the state 3/state 4 transition (Figure 1D). This is due to the presence of small amounts of oxaloacetate and the slow return of the MDH equilibrium (Hagedorn et al., 2004).
The changes in total, bound, and free NADH during succinate oxidation are shown in Figure 2. Here, the same pattern was observed as for malate oxidation except that the changes in NADH during the state 3/state 4 transitions were more rapid, reflecting the fact that no oxaloacetate was produced and that MDH was not involved in the reactions (Hagedorn et al., 2004
The disappearance of the fluorescence signal coming from bound NADH can be due to two processes: (1) Its conversion to NAD+, which is not fluorescent and hence not detectable by our method and/or (2) its release from the binding site of an enzyme. With the increased respiration rate in state 3, more NADH is oxidized, leading to a lower steady state level of bound NADH (Hagedorn et al., 2004 ). Under equilibrium conditions, the free NADH concentration would readjust accordingly (i.e., decrease), but this does not happen. Surprisingly, the concentration of free NADH remains relatively constant in state 3 (Figures 1D and 2B), leading to a large increase in the proportion of free NADH (Figures 1E and 2C). We have attempted to explain this phenomenon by modeling NADH and NAD+ binding to mitochondrial proteins.
Modeling
Assuming that states 3 and 4 are both in steady state, we wish to determine sets of parameters for each state that reproduce the data in Table 3—the most interesting aspect being the slight increase in the amount of free NADH from state 4 to state 3.
There are five parameters: n, e, KC, KNADH, and
This leaves us with KNADH and
In case 1, keeping both KNADH and
Thus, we conclude that a change in the binding properties of NAD+, as calculated by the steady state model defined in Equations 1 to 5 (see Methods), cannot account for the experimental results. However, the data can by reproduced by changing the average binding of NADH to proteins in the matrix and on the inner surface of the inner mitochondrial membrane. We estimated that KNADH should decrease 2.5 to 3 times in state 3 compared with state 4.
We have here shown that plant mitochondria contain two pools of NADH, free NADH, and protein-bound NADH. Bound NADH dominates in the absence of ADP (states 2 and 4), whereas free NADH is highest in the presence of ADP (state 3). However, the concentration of free NADH, the parameter determining the degree of interaction with enzymes, is surprisingly constant. Modeling shows that this observation can be explained by a decrease in the average binding of NADH to proteins by a factor of 2.5 to 3, whereas changes in NAD+ binding cannot. The bound NADH is probably predominantly associated with the active site of oxidoreductase enzymes using it as a coenzyme. The mitochondrial matrix contains a number of abundant dehydrogenases, namely, pyruvate dehydrogenase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, MDH, and malic enzyme, as well as many less-abundant NADH binding enzymes. The total concentration of binding sites is probably in the low millimolar range similar to the concentration of NADH (Møller, 2001 ). Binding to other proteins, such as that observed for BSA (see Methods), which is probably nonspecific, cannot be excluded, but there is no evidence for such binding. There is however a fundamental difference between NADH bound to the active site of enzymes and nonspecifically bound NADH: The former is directly involved in metabolic processes (it has been caught in the act), whereas the latter constitutes an inactive pool.
The observation that free NADH in the mitochondrial matrix is maintained at a relatively constant level irrespective of respiratory state leads to two important conclusions: (1) The concentration of free NADH does not play a regulatory role in respiration. If it did, such a crude regulation would affect far too many processes simultaneously. Hence, it seems reasonable that the pool of free NADH is in homeostasis, while mitochondrial processes are regulated by other more specific mechanisms. (2) There is a specific mechanism for maintaining NADH homeostasis. Assuming that a steady state condition is reached at the end of each state, the binding affinity of the mitochondrial protein matrix must be dynamically adjusted during the respiratory cycle to keep the free NADH constant. Modeling shows that free NADH homeostasis is most likely due to changes in the binding properties of NADH, rather than NAD+. According to our calculations, the average binding constant of NADH to mitochondrial proteins in state 3 should decrease by a factor of 2.5 to 3 relative to binding in state 4. One factor that can influence the enzyme–NADH interaction is the competitive binding of other metabolites or accumulation of reaction products. Another possible mechanism would be protein phosphorylation, which affects a number of the most active and abundant NAD(H) binding mitochondrial enzymes (Bykova et al., 2003
Here, we have demonstrated that potato tuber mitochondria contain an appreciable and fairly stable amount of free NADH in different metabolic states. Our data are in contrast with Wakita et al. (1995)
Since 20% (state 4) to 60% (state 3 with succinate) of the total NADH is free in potato tuber mitochondria (Figures 1E and 2C), it means that the concentration of free NADH in the matrix is 100 to 150 µM or 50 to 75 µM, assuming a matrix volume of 1 or 2 µL/mg mitochondrial protein, respectively. One consequence of this is that because the concentration of free NADH is >3 times higher than the Km (NADH) for both complex I and the rotenone-insensitive NADH dehydrogenase on the inner surface of the inner membrane of potato tuber mitochondria (Rasmusson and Møller, 1991 It will be interesting to apply the method for determining free and bound NADH developed here to isolated pea (Pisum sativum) leaf mitochondria, which differ in the following important aspects from potato tuber mitochondria.
Pea leaf mitochondria participate in photorespiration and therefore contain a very high concentration of the glycine decarboxylase complex ( The method described here will also be applicable to other isolated organelles, intact cells, and perhaps even tissues. Knowledge about the proportion of free NAD(P)H in living tissues is crucial for interpreting their metabolic behavior. Since NAD(P)H is involved in detoxification of reactive oxygen species, the method might also provide a sensitive assay for oxidative stress.
Materials NADH, yeast ADH, fat-free BSA, and other chemicals were purchased from Sigma-Aldrich and were of the highest grade available.
Mitochondria were isolated from potato tubers (Solanum tuberosum cv Bintje) bought in the local market by differential centrifugation followed by Percoll density gradient centrifugation essentially as described by Struglics et al. (1993)
Extraction and Quantification of Total Mitochondrial NAD(H)
Binding of NADH to Model Proteins
The calorimetric measurements were conducted with an MCS-ITC isothermal titration calorimeter (MicroCal) at 25°C. NADH and/or proteins were dissolved in 5 mM HEPES-KOH, pH 7.5. Concentrations of the proteins and NADH were determined spectrophotometrically using the published extinction coefficients (
The protein solution, typically between 2 and 10 mg/mL, was loaded into the sample cell (Vcell = 1.3 mL) and titrated with a concentrated solution of NADH. The experiments were designed so that the amount of injected ligand at the end of a titration experiment was twice the number of potential binding sites of the protein. The results were fitted to a model with one set of independent binding sites. An example of a titration experiment for ADH is shown in Figure 4. The binding parameters were obtained by floating association constant (ka), binding enthalpy (
The studied dehydrogenases are believed to be dimers. However, most of the obtained numbers were consistently lower than two binding sites per dimer. This usually happens when some of the binding sites are inactive or occupied by another ligand. Our inability to obtain exactly two binding sites per dimer was probably due to the presence of some initial amount of NADH. This problem can be circumvented by the assumption that the number of available binding sites is smaller than the actual concentration of the protein. Adjusting down the concentration of the macromolecule gives the right number of binding sites but has no influence on the results of the fitting for the association constant. Therefore, the ka and n obtained from ITC could be used directly in the treatment of the model system fluorescence data.
Spectra of Free and Bound NADH We selected yeast ADH as the model protein with an intermediate between specific and nonspecific binding properties for NADH. This model system was routinely tested prior to the beginning of the experiments with the mitochondria.
The spectrum of free NADH was measured directly on the solutions of pure NADH at different concentrations. The linearity of the fluorescence signal with the concentration of NADH was ensured using low concentrations (i.e., 1 to 10 µM). Using the binding parameters obtained from ITC, the concentrations of free and bound NADH were calculated for the three standard mixtures of NADH and ADH (Table 5, Figure 5A). These systems were designed to contain the same amount of total NAD(H) as our biological system, calculated with the assumption that the isolated mitochondria from potato tubers contain 1 to 2 nmol NAD(H) (mg protein)–1 (Agius et al., 2001
Decompositions of Mitochondrial Fluorescence Spectra Decomposition was based on the notion that an experimental emission spectrum can be written as a linear superposition of the two emission spectra of free and bound NAD(P)H. Moreover, it was assumed that the emission spectrum of NAD(P)H bound to any protein closely resembles the emission spectrum of NADH bound to ADH. Specifically, spectra are nonnegative functions of frequency on the experimentally accessible frequency interval. Squared-integrable real functions on this interval form a vector space. The two emission spectra of free and bound NAD(P)H are two linearly independent vectors in this space. The experimental emission spectrum is a third vector in the same space and lies in the two-dimensional subspace spanned by the first two vectors if the experimental emission spectrum originates in free and bound NAD(P)H. Least-squares fitting of a linear combination of the first two spectra to the third vector (the experimental spectrum) yields the linear combination of the first two that differs the least from the latter. If the difference vanishes, one has a perfect decomposition. If it does not, the magnitude of the residual gives an estimate of the size of the error committed by claiming that the decomposition represents the experimental emission spectrum.
In practice, we represented the spectra by their values at 1100 frequency values. Then, a vector in the abstract function space is represented by an ordinary vector with 1100 real elements, and decomposition becomes matrix algebra: Emission spectra of the species free NADH and NADH bound to ADH (arranged in an 1100 x 2 matrix B) were calculated from emission spectra of n standard mixtures of NADH and ADH (in a 1100 x n matrix S) using the 2 x n matrix C of the NADH species concentrations in the standard mixtures as follows:
The species concentrations in C were calculated from total amounts of NADH and ADH and the binding constant of NADH and number of NADH binding sites on ADH obtained by ITC.
Concentrations of the NADH species in a series of m experimental emission spectra (arranged in an 1100 x m matrix Z) were then calculated by writing each column in Z as a linear combination of the two columns in B and a vector orthogonal to those two columns. Doing this for all m spectra in Z, there are m orthogonal vectors. These can be arranged in a residual matrix R, so in matrix form one has the following:
This decomposition of experimental spectra in Z after the two spectra in B is equivalent with least-squares fitting of a linear superposition of the two spectra in B to each of the spectra in Z. The parameters fitted are the elements of D, and their resulting values are those given in the identity for D above. The quality of this fit was evaluated by inspection of the residual spectra arranged in the 1100 x m matrix R as follows:
Fluorescence Measurements
There are two advantages to the use of a pulsed excitation regime: (1) Bleaching is reduced, and (2) it allows collection of time-gated data in addition to the steady state spectra. Expanding the method initially introduced by Paul and Schneckenburger (1996) , we made a series of measurements of NADH fluorescence lifetimes in different metabolic states. The measurements of fluorescence decays have an advantage over the steady state measurements because the fluorescence lifetime of bound NADH is an order of magnitude longer than that of a free NADH (see Introduction). Consistent with this, our preliminary results indicated that the observed fluorescence of potato mitochondria has a longer lifetime in those states that have an increased proportion of bound NADH. Quantification of this qualitative result, however, requires a rigorous mathematical treatment not attempted in this study and points toward further possible improvement of our method.
Calibration of the wavelength scale of the spectrometer was conducted prior to the experiments. Correction of the fluorescence emission intensity to the instrument response function was performed only once to ensure the correct location and shape of the NADH fluorescence. This is shown in Figure 7, and the excitation peak in the corrected spectrum was shifted
Modeling
At steady state, Reactions a to c can be written as the following equations:
 , and KC are the equilibrium constants. Reactions a to c imply that the total concentration of binding sites (e) and the total concentration of NAD(H) (n) are constant:
Equations 1 to 5 constitute five equations, with five parameters (KNADH,
Combination of Equations 1 to 5 yields the equations from which [NADH] (or [E]) can be calculated:
The solution for [NADH] is as follows:
The other concentrations follow from this.
We thank Ina Blom for excellent technical assistance, Peter Westh for loan of the ITC instrument, Søren K. Rasmussen and Steen G. Hanson for continuous support, and the following organizations and agencies for economic support: Center for Biomedical Optics and CIRIUS, Danish Ministry of Education, to J.G., Danish Natural Science Research Council to I.M.M., and Risø National Laboratory to M.R.K., P.E.A., and I.M.M.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Ian M. Møller (imm{at}kvl.dk). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.039354. Received November 9, 2005; Revision received December 22, 2005. accepted January 9, 2006.
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ABSTRACT
INTRODUCTION
2 < 1. 
340 nm [NADH] = 6.22 mM–1 cm–1 [Putnam, 1975]; 
H), and the number of binding sites (n). The obtained values for dehydrogenases (
