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Keywords:

  • Respiratory mutant;
  • Screening;
  • Mitochondria;
  • Triphenyltetrazolium chloride;
  • Cytochrome oxidase

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgments
  8. References

The inability of cells and microorganisms to reduce the colourless electron acceptor triphenyltetrazolium chloride (TTC) to a red formazan precipitate is commonly used as a means of screening for cells that have a dysfunctional respiratory chain. The site of reduction of TTC is often stated to be at the level of cytochrome c oxidase where it is assumed to compete with oxygen for reducing equivalents. However, we show here that TTC is reduced not by cytochrome c oxidase but instead by dehydrogenases, particularly complex I, probably by accepting electrons directly from low potential cofactors. The reduction rate is fastest in coupled membranes because of accumulation in the matrix of the positively charged TTC+ cation. However, the initial product of TTC reduction is rapidly reoxidised by molecular oxygen, so that generation of the stable red formazan product from this intermediate occurs only under strictly anaerobic conditions. Colonies of mutants defective in cytochrome oxidase do not generate sufficiently anaerobic conditions to allow the intermediate to form the stable red formazan. This revision of the mode of interaction of TTC with respiratory chains has implications for the types of respiratory-defective mutants that might be detected by TTC screening.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgments
  8. References

The inability of cells and microorganisms to reduce the colourless electron acceptor TTC (triphenyltetrazolium chloride) to a red formazan precipitate has been used as a means of screening for cells that have dysfunctional respiratory chains. The screen has been used successfully to identify a range of cytochrome oxidase-defective mutants of bacteria, yeasts and Chlamydomonas reinhardtii[1–4]. It is often assumed [3,5], following an early comprehensive study of a range of tetrazolium salts [6], that TTC competes with oxygen for reducing equivalents from cytochrome c oxidase, hence accounting for its action as a screen for oxidase mutants and for the observed inhibition of formazan formation by inhibitors of cytochrome c oxidase and of complex III [6].

However, it is well known that the haem a3/CuB binuclear centre of cytochrome c oxidase is accessible only to molecules much smaller than TTC [7] and, furthermore, the midpoint potential of TTC (the midpoint potential at pH 7, Em7, of which has been variously reported as −83, −240 or −415 mV versus SHE (the standard hydrogen electrode) [6]) is too low to be reducible by the binuclear centre at a reasonable rate. The data reported here with respiratory chains from a variety of organisms confirm that TTC is not reduced by cytochrome c oxidase but instead interacts with dehydrogenases, particularly complex I, probably by accepting electrons directly from the low potential cofactors. Reduction of TTC by dehydrogenases has already been noted [8,9], but has not been equated with its ability to act as a screen for cytochrome c oxidase mutants. We show here that the initial product of TTC reduction is rapidly reoxidised by molecular oxygen (cf. [10]), so that generation of the stable red formazan product occurs only under strictly anaerobic conditions. Mutants defective in cytochrome oxidase do not generate sufficiently anaerobic conditions in order to allow formazan production and it is for this reason that it can be used successfully as a screen for such mutants.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgments
  8. References

2.1Preparation of mitochondrial membranes

Keilin–Hartree particles from beef heart were prepared as described in [11] and stored at 77 K until required. Protein concentrations were determined using a modified Biuret method in which 0.5 mM H2O2 and 0.5% sodium cholate are added during sample preparation [12].

Mitochondria were isolated from pea leaves (Pisum sativum L. cv. Massey Gem) and purified on a Percoll gradient as described by Day et al. [13] and submitochondrial particles according to [14]. Protein was assayed as described in [15].

2.2Growth of C. reinhardtii

The Chlamydomonas chlorophyll-minus strain was a gift from W.-Y. Wang (University of Iowa) and carries two nuclear mutations, pc-1 and y-7, that block chlorophyll biosynthesis by the light-dependent (pc-1) and light-independent (y-7) pathways [16]. As a consequence, the mutant fails to assemble any of the chlorophyll-containing complexes of the thylakoid membrane. The mutant was grown in TAP medium [17] in complete darkness at 25°C to a cell density of ∼2×106 ml−1. Cells were concentrated by centrifugation immediately before use.

2.3Formazan production and oxygen consumption

The rate of generation of formazan was monitored with a double beam spectrophotometer with the wavelength pair 570–620 nm and was quantitated using a gravimetrically determined extinction coefficient, ?, of 3.6 mM−1 cm−1 or at 500 nm alone (?=5.6 mM−1 cm−1). Oxygen consumption was measured polarographically with a Clark-type oxygen electrode. Reaction medium in all cases was 250 mM sucrose, 10 mM TES, 10 mM potassium phosphate and 5 mM MgCl2 at pH 7.2 and room temperature.

2.4Cytochrome c redox state in intact Chlamydomonas cells

The redox state of cytochrome c was used as a means of following aerobic/anaerobic transitions in intact cells of C. reinhardtii in the presence of TTC. This was achieved by cyclic monitoring of the wavelengths 542, 550, 558, 570 and 620 nm, from which TTC reduction and cytochrome c redox changes were deconvoluted as 570–620 nm and 550−(542+558)/2 nm, respectively. This triple wavelength assay for cytochrome c largely removed interference from the overlapping spectral changes due to TTC reduction.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgments
  8. References

3.1Electrochemical properties of TTC

Because of some uncertainty in the published electrochemical properties of TTC, the voltammetric behaviour of TTC was examined using conventional three-electrode cyclic voltammetry. A 3-mm diameter platinum surface was used as a working electrode, together with a platinum spade counter electrode and a 3-M KCl silver/silver chloride reference. Potentials were calibrated using benzyl viologen (Em7=−360 mV) as a standard. For 5 mM TTC in mitochondrial reaction medium, negative sweep at 100 mV s−1 of an unstirred anaerobic solution produced a reductive half-wave at −295 mV versus SHE at pH 7.2. Subsequent positive sweep did not produce an oxidative wave, indicating the irreversibility of the reduction process. Furthermore, each subsequent cycle showed reductive waves of decreasing amplitude as the surface of the electrode rapidly became coated with the insoluble red formazan product. A repeat of the measurement under aerobic conditions produced the same result, except that the reductive wave was larger and more cycles were required before the wave disappeared. It is concluded that the effective potential required to reduce TTC is in the −295-mV region at pH 7.2 and that an initial reduced product, presumably a one-electron state, can be reoxidised by oxygen or can accept a second electron to produce the irreversible formazan precipitate.

3.2Formazan formation requires anaerobic conditions

Fig. 1 shows the rate of formation of formazan from TTC in coupled pea leaf mitochondria respiring aerobically with glycine as respiratory substrate [18], monitored spectrophotometrically at 570–620 nm. The rate was extremely slow until anaerobiosis had been achieved at which point the reduction rate increased dramatically to a steady rate of 22 nmol formazan produced per mg protein per minute. Under aerobic conditions neither ADP nor cyanide (not shown) increased the rate significantly.

image

Figure 1. Formazan formation by coupled pea leaf mitochondria. Pea leaf mitochondria were resuspended to 0.29 mg ml−1 protein in an aerobic reaction medium at pH 7.2 and 25°C and containing 0.3 mM ATP and 0.1 mM TTC. Respiration was initiated with 10 mM glycine and ADP was added to 1 mM. Anaerobiosis occurred after approx. 10 min under these conditions. Formazan production was monitored at 570–620 nm and rates are given in nmol (mg protein)−1 min−1.

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Analogous behaviour was found with bovine heart Keilin–Hartree mitochondrial particles. Under aerobic conditions and with succinate as substrate, the rate of formation of formazan from TTC was negligible until anaerobiosis had occurred (Fig. 2). Subsequent addition of NADH, which is oxidised by complex I, increased the rate 5-fold. Lack of formazan formation in aerobic conditions was further demonstrated by introduction of oxygen by stirring: each time this caused a complete cessation of formazan formation until anaerobiosis had again been established. Note, however (see below), that the rate after the oxygen addition was always lower than the previous rate even though the anaerobic rate itself was fairly constant. Furthermore, added oxygen did not reoxidise the formazan that had already formed, confirming that formazan itself is not autoxidisable.

image

Figure 2. Formazan formation by bovine Keilin–Hartree mitochondrial particles. Keilin–Hartree particles were resuspended to approx. 2 mg ml−1 protein in 50 mM potassium phosphate buffer at pH 8.0 and room temperature and containing 400 μM TTC. The rate of formazan formation was monitored at 500 nm. Respiration was initiated with 10 mM potassium succinate. Anaerobiosis, indicated by the arrow, occurred after approx. 1 min. Subsequently, 1 mM NADH and two aliquots of air were added where indicated.

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In order to investigate further the rate at which TTC reacted in the aerobic state, oxygen uptake of pea leaf mitochondria respiring on malate/glutamate was monitored (malate oxidation provides a supply of NADH and glutamate removes the oxaloacetate product so that a steady rate is maintained). Addition of cyanide (Fig. 3) caused a dramatic decrease in respiration rate, as expected since pea leaf mitochondria do not contain an alterative oxidase [19]. Subsequent addition of 0.5 mM TTC resulted in a slow but measurable additional rate of oxygen consumption, which slowly decreased over the following 10 min. The increase in oxygen consumption caused by TTC addition (7 nmol mg−1 min−1) was similar to the rate of TTC reduction observed anaerobically when the same concentration of TTC was added to an equivalent sample that had been allowed to become anaerobic in the absence of cyanide.

image

Figure 3. Oxygen consumption induced by TTC in pea leaf mitochondria. Pea leaf mitochondria were resuspended to 0.29 mg ml−1 protein in reaction medium at pH 7.2 and 25°C. Additions were 10 mM glutamate/10 mM malate, 0.5 mM HCN and 0.5 mM TTC. Numbers along the trace are nmol O2 consumed (mg protein)−1 min−1.

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From the above, it is clear that TTC accepts electrons from the respiratory chain in the aerobic state but, instead of producing formazan, is rapidly reoxidised by oxygen. However, formazan itself does not autoxidise (see Fig. 2 and above). Hence, formazan formation (which requires two electrons [3]) must occur via an unstable autoxidisable intermediate, most likely the one-electron reduced form.

3.3Formazan production is faster in coupled membranes

Fig. 4 shows formazan production after addition of TTC to pea leaf mitochondria that had already become anaerobic in the presence of glycine and ADP. A rapid rate of formazan formation occurred with only a small lag due to the oxygen that was introduced along with the TTC. The rate was substantially faster than the anaerobic rate observed when TTC had been added whilst oxygen was being consumed (cf. Fig. 1). Addition of the uncoupler FCCP (carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, 0.13 μM) or DNP (dinitrophenol, 50 μM, not shown) caused a drastic decrease in rate. However, addition of further TTC increased the uncoupler-resistant rate by a factor roughly equivalent to the increased concentration of TTC. It is concluded that the formazan formation rate is proportional to the concentration of TTC at the site at which it is reduced and that this site is within the mitochondrial matrix space. In coupled membranes the membrane potential causes the positively charged TTC cation to accumulate within the mitochondrial matrix, leading to a higher rate.

image

Figure 4. Formazan formation by pea leaf mitochondria respiring with different substrates. Pea leaf mitochondria were resuspended to 0.29 mg ml−1 protein in an aerobic reaction medium at pH 7.2 and 25°C and containing 1 mM ADP and 0.3 mM ATP. Formazan formation was monitored at 570–620 nm. Respiration was initiated with 10 mM glycine, 10 mM glutamate/10 mM malate, 10 mM succinate or 1 mM NADH. TTC additions were as indicated to 0.1 or 0.5 mM and FCCP was added to 1.3 μM.

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3.4The sites of TTC reduction in mitochondria

Rates of reduction of TTC by pea leaf mitochondria respiring with glycine, malate/glutamate, succinate or externally added NADH are compared in Fig. 4 and Table 1. In all cases the TTC was added only after the sample had become anaerobic so that maximum rates were assessed. The highest rates of TTC reduction under coupled conditions were observed with glycine and malate/glutamate, both of which can donate reducing equivalents to complex I and the alternative internal NADH dehydrogenase(s) (NDH-2). The rate of TTC reduction was slower with succinate and the rate with added NADH (which is oxidised by the externally facing NADH dehydrogenase(s), NDH-3) was slowest of all and also decreased with time. Uncoupler (0.13 μM FCCP) dramatically inhibited TTC reduction by all substrates. However, a subsequent increase of the amount of TTC to 0.5 mM stimulated the FCCP-inhibited rate by a factor of approx. 5 (i.e. the same as the increase in TTC concentration).

Table 1.  Effects of FCCP on formazan production by various substrates in pea leaf mitochondria
SubstrateRate of formazan production (nmol (mg protein)−1 min−1)
 100 μM TTC/coupled500 μM TTC+0.13 μM FCCP
  1. Data of Fig. 4 were quantitated with an extinction coefficient of 3.6 mM−1 cm−1. Rates were measured immediately before FCCP addition (i.e. anaerobic and coupled with 0.1 mM TTC) or after the second addition of TTC (i.e. anaerobic, uncoupled at 0.5 mM TTC). Data are rates from a single mitochondrial sample used in Fig. 4, but are representative of typical rates obtained with five different preparations.

Glycine6435
Malate/glutamate4316
Succinate187
NADHexternal56

These observations are interpreted to indicate that TTC is reduced by the dehydrogenases with rates for complex I (and/or NDH-2)>succinate dehydrogenase>NDH-3. Since the sites of reduction of TTC by all dehydrogenases except NDH-3 are within the matrix space, rates are fastest in the absence of the uncoupler FCCP, but they decrease dramatically when FCCP is added since it dissipates the membrane potential and hence decreases the matrix TTC concentration. Addition of further TTC increases the rate by increasing the effective TTC concentration at the site of reaction.

The rate of formazan production by external NADH under coupled anaerobic conditions was slowest and also decreased with time. An inhibitory effect of FCCP was also observed, but was much smaller than that for the other substrates. Reduction of TTC by NDH-3 should occur at the external face of the inner membrane, in contrast to reduction by complex I and succinate dehydrogenase which occurs within the matrix space. Hence, the small FCCP effect observed for the NADH-supported rate may indicate that part of the reduction pathway involves a reversed electron transfer to succinate dehydrogenase (and, possibly, complex I and NDH-2) via the quinone pool. As for the other substrates, the uncoupled rate increased in proportion to the TTC added.

The rate of formation of formazan with glycine under anaerobic uncoupled conditions was not significantly inhibited by myxothiazol, antimycin A (inhibitors of complex III), cyanide (an inhibitor of cytochrome oxidase) or rotenone (an inhibitor of the ubiquinone reduction site of complex I). The uncoupled succinate-supported rate was also not inhibited by these compounds, nor by carboxin (an inhibitor of the ubiquinone reduction site of complex II). It is therefore concluded that TTC can accept reducing equivalents directly from the low potential cofactors of the dehydrogenases, most likely by interaction via the donor (NADH or succinate) sites themselves, rather than via the ubiquinone-reactive sites, a conclusion consistent with the hydrophilic nature of TTC and its rather low redox potential [6]. It was also noted that the rate observed with succinate under coupled anaerobic conditions was not inhibited by rotenone (not shown), ruling out a pathway mediated by reversed electron flow through complex I and demonstrating that succinate dehydrogenase can donate electrons directly to TTC, though at a rate slower than that of complex I. This conclusion was further corroborated by the finding that the rate of TTC reduction by pea leaf mitochondria under coupled anaerobic conditions supported by succinate plus glycine (82 nmol mg−1 min−1) was the sum of the two rates supported by each substrate on its own (23 and 56 nmol mg−1 min−1, respectively).

In order to assess the relative rates of formazan formation by complex I and NDH-2, a separate experiment was performed with submitochondrial particles that had been prepared from soybean cotyledons. These particles are primarily inside out; added NADH is oxidised by both complex I and NDH-2 at roughly equivalent rates whereas the substrate analogue deamino-NADH is oxidised only by complex I [20]. However, the rate of anaerobic reduction of 0.5 mM TTC by 1 mM NADH was found to be essentially the same as that with 1 mM deamino-NADH. Furthermore, addition of NADH to a sample that was reducing TTC in the presence of deamino-NADH stimulated the rate by less than 20%. Hence, it can be concluded that complex I reduces TTC to formazan much more readily than does NDH-2.

3.5TTC reduction in intact cells of C. reinhardtii

In order to confirm that TTC interacts with the respiratory chain of microorganisms in the same way as with isolated eucaryotic mitochondria, experiments were performed to examine the TTC reduction pathway by intact cells of C. reinhardtii. In order to be able to use sufficient cell mass to detect TTC reduction on a reasonable timescale, it was necessary to use a mutant strain impaired in chlorophyll biosynthesis [16] since the chlorophyll otherwise absorbed too strongly to allow assay of TTC and cytochrome c. The aerobic/anaerobic transition can be monitored by following the redox state of cytochrome c, which is largely oxidised in the aerobic state and becomes reduced on anaerobiosis. The cells had already become anaerobic at the start of the traces in Fig. 5 so that the cytochrome c was reduced. Mixing oxygen into the anaerobic sample led to a reoxidation of cytochrome c (shown as a downward deflection in Fig. 5, bottom). Addition of TTC to the aerobic sample did not result in a significant rate of formazan production. However, restoration of anaerobiosis (as monitored by re-reduction of cytochrome c) was associated with an onset of TTC reduction, which appeared as a burst phase followed by a slower steady state rate. The burst probably arises because the mitochondria still have a membrane potential at the point of anaerobiosis, but this soon decreases so that the concentration of TTC within the mitochondria diminishes, resembling the inhibitory effect of uncoupler on TTC reduction in isolated mitochondria described above. Addition of cyanide did not inhibit the formazan production rate, which again showed a short burst due to the small amount of oxygen added with the cyanide which transiently restored the membrane potential (Fig. 5). The anaerobic rate of TTC reduction was also insensitive to 5 μM myxothiazol and to 5 μM antimycin A (data not shown). Since these compounds inhibit cytochrome oxidase directly (cyanide) or inhibit electron donation to cytochrome oxidase (myxothiazol and antimycin A), the data show that cytochrome oxidase is not the reductant of TTC and that TTC must instead be interacting with the more reducing part of the respiratory chain, presumably with dehydrogenases. This, together with the requirement again for anaerobic conditions, indicates that the mode of action of TTC is the same as that seen above with mitochondrial membranes, as expected from the very close similarity of the Chlamydomonas and higher plant respiratory chains.

image

Figure 5. Simultaneous measurement of formazan formation and cytochrome c redox state in whole cells of a chlorophyll-free strain of C. reinhardtii. Cells of a chlorophyll-free strain of C. reinhardtii were resuspended in TAP medium to around 100 mg ml−1 wet weight. The wavelengths 542, 550, 558, 570 and 620 nm were monitored, from which TTC reduction and cytochrome c redox changes were deconvoluted as 570–620 nm and 550−(542+558)/2 nm, respectively. Where indicated, additions were oxygen, 2 mM TTC, or 1 mM cyanide. Formazan production and cytochrome c reduction both result in increasing (upward) absorbance changes at the wavelengths used.

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4Conclusions

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgments
  8. References

We show here that TTC is reduced in a concentration-dependent manner by the mitochondrial dehydrogenases, most rapidly by complex I and, more slowly, by other dehydrogenases. The mechanism is the same in mitochondria from diverse sources and we have also established that the mechanism is the same for its interaction with the respiratory chain of Escherichia coli (unpublished data). Under aerobic conditions no red formazan develops, and a slow inhibition of respiratory chain components is observed, probably caused by the superoxide/peroxide products of autoxidation [10]. Only under strictly anaerobic conditions does the unstable reduced intermediate go on to form the red stable formazan at reasonable rates. Cytochrome c oxidase has a very high affinity for oxygen (around 0.1 μM [21,22]) so that cell colonies growing on agar plates and utilising respiration via cytochrome c oxidase are anaerobic in the colony centre. Hence, such cells will accumulate the red formazan when overlaid with TTC as described [3]. Dysfunction of the cytochrome pathway will preclude sufficiently anaerobic conditions and so prevents formazan formation from the unstable intermediate which instead autoxidises back to TTC. Hence, lack of formazan formation does indeed act as a screen for cytochrome pathway mutants, even although the TTC itself accepts electrons from the dehydrogenases. The need for anaerobic conditions also explains the original observations of an inhibition of formazan formation by mitochondria on addition of antimycin A or cyanide [6] since these reagents will prevent anaerobiosis by blocking the cytochrome chain.

Many types of cells, including those of C. reinhardtii, possess an alternative respiratory oxidase that is linked to the respiratory chain at the level of ubiquinone and also reduces oxygen to water [23]. Nevertheless, the TTC overlay technique can still be used successfully to screen for cytochrome chain mutants [2,4]. Hence it is clear that the anaerobic conditions required for formazan formation cannot be attained by operation of the alternative oxidase alone, consistent with its known lower affinity for oxygen [21].

This revised explanation of the mechanism of TTC action has consequences for the types of mutants that might exhibit a ‘formazan-minus’ phenotype. Its use to screen for mutants defective in the cytochrome pathway is already well established. However, the data here show that the fastest rate of formazan formation under anaerobic conditions occurs with substrates of complex I and with well-coupled membranes. Hence, in some instances the phenotype might arise because of a defective complex I and/or membranes that are unable to maintain a reasonable protonmotive force. Such mutants should be discernable by repeating the screen under anaerobic conditions where the phenotype would still occur.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgments
  8. References

We are grateful for the financial support of an Australian Research Council IREX grant (to P.R.R. and J.T.W.) and to Jonathan Ramsey for expert technical assistance.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgments
  8. References
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