The sites of superoxide anion generation in higher plant mitochondria

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 188, No. 1, May, pp. 206-213, 1978

The Sites of Superoxide PETER Johnson

Research

Foundation,

Anion Generation

R. RICH

AND

WALTER

in Higher D. BONNER,

Department of Biochemistry and Biophysics, Philadelphia, Pennsylvania 19174

Received November

Plant Mitochondria’

23, 1977; revised January

JR. University

of Pennsylvania,

27, 1978

A variety of higher plant mitochondria and submitochondrial particles with varying degrees of cyanide insensitivity have been examined for their possible superoxide anion generating capacity. It was found that neither the cytochrome oxidase nor the alternative oxidase pathways produced significant quantities of superoxide anions. All preparations examined generated superoxide anions to a small extent with NADH as respiratory substrate, but almost negligibly with succinate as respiratory substrate. A component of the NADH-supported activity was insensitive to cyanide, antimycin A, and salicylhydroxamic acid. Hence most of this activity is attributed to direct reduction of oxygen by the flavoprotein NADH dehydrogenases. The remainder may be caused by oxygen red&ion in the ubiquinone-cytochrome b region of the chain. In some plant mitochondria and submitochondrial particles, a contaminating tyrosinase activity, which can catalyze the oxidation of epinephrine by molecular oxygen, causes a very large interference in superoxide anion determinations. Methods of distinguishing and measuring these various activities are discussed.

The water

reduction requires

of molecular four

reducing

02 + 4e- + 4H+ +

oxygen

to

equivalents:

2HzO.

Since the midpoint potential of the 02/H20 couple at pH 7.0 is around +800 mV (l), the overall reaction is thermodynamically very favorable. Kinetically, however, the reaction is extremely sluggish and a catalyst is required for a significant rate of reaction to occur. Intermediary stages of oxygen reduction are also possible, these being the superoxide (02J and peroxide (0~‘~) states. Many oxidative enzymes are known which can catalyze this reduction of molecular oxygen, and the product of the reduction may be superoxide, peroxide, or water. Mechanistically it is of great, interest to determine the final product of an enzymatic reduction of oxygen, since this may provide information on the number and types of redox centers involved in the reaction. It is already known that the mitochondrial cytochrome oxidase catalyzes the four-electron reduction of oxygen to water and no ’ Abbreviation sulfonic acid.

used: Mops, I-morpholinepropane206

0003-9861/78/1881-0206$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

free intermediary products can be detected (2). It is also well documented that oxygen may be reduced to superoxide (and hence to peroxide via superoxide dismutase) in the ubiquinone-cytochrome b region of the respiratory chain (3-5), probably at the level of an unstable ubisemiquinone species (6). In higher plants, an “alternative oxidase” (7) may reduce oxygen to hydrogen peroxide (8), although studies of the product of this reaction have been hampered by the contaminating activities of catalase and peroxidase in the preparations (8). In this report, an attempt has been made to define the possible sites of oxygen reduction by higher plant mitochondria, so that we have a framework on which to base future studies. The situation is rather more complex than in mammalian systems, since we have the added parameters of extra oxidases and at least one extra NADH dehydrogenase (9). It is shown that, besides cytochrome oxidase and the alternative oxidase, oxygen reduction can occur at the level of the NADH dehydrogenases and possibly also at the ubiquinone-cytochrome b segment. Only these latter two

SUPEROXIDE

GENERATION

processes produce superoxide, as measured by superoxide dismutase-sensitive epinephrine oxidation. A further apparent route of superoxide anion generation in some higher plant mitochondria is caused by a contaminating tyrosinase activity, which can catalyze the oxidation of epinephrine by molecular oxygen in a salicylhydroxamic acidand cyanide-sensitive manner. A system, based upon inhibitor sensitivities of these routes, has been devised so that the activities of each may be detected and distinguished. MATERIALS

AND

METHODS

Preparation of mitochondria and submitochondrial particles. Mung bean hypocotyls were excised from plants grown from seeds in a darkroom for 5 days at 28’C and 60% relative humidity. Potato tubers (Solarium tuberosum) and tulip bulbs ( Tulipa gesnerana var. Darwin) were purchased locally. Symplocarpus foetidus spadices were collected from swamp areas in Pennsylvania and Arum maculatum inflorescences were collected in Cambridgeshire, England. Mitochondria were prepared as described by Bonner (IO). In the case of S. foetidus spadices, the EDTA concentration was increased to 2 mM in the homogenization medium and bovine serum- albumin was increased to 0.5% (w/v) in both the homogenization and resuspension media. Submitochondrial particles were prepared as described by Rich and Bonner (11) unless otherwise stated. Superoxide anion assay. Superoxide anion generation was assayed by monitoring the rate of formation of adrenochrome from epinephrine at 485 minus 575 nm (E = 2.96 mMm' cm-‘) (6, 12, 13) with a doublebeam spectrophotometer. The medium used was 30 mM Tris-Mops at pH 8.0 with 1 mM epinephrine added. Tyrosinase assay. Tyrosinase was assayed in 50 mM sodium citrate at pH 5.6 and 25°C. The substrate was either 1 mM epinephrine, 0.1 M pyrogallol, or 3.3 mM I,-tyrosine. The initial rates of oxygen consumption were used as a measure of enzyme activity. Oxygen consumption. The oxygen consumption of mitochondria was measured with a Clark-type oxygen electrode and with a medium containing 0.3 M mannitol, 10 mM KCI, 5 mM MgCi,, and 10 mM potassium phosphate at pH 7.2 and 25’C. Others. Protein was measured by the method of Lowry et al. (14) with crystalline bovine serum albumin as a standard. Partially purified tyrosinase (from mushroom, EC 1.14.18.1) and superoxide dismutase (from bovine blood, EC 1.15.1.1) were purchased from Sigma Chemical Co.

IN PLANT

207

MITOCHONDRIA RESULTS

Tyrosinase

Oxidation

of Epinephrine

It was observed that, when epinephrine was added to potato mitochondria and submitochondrial particles in the presence of oxygen, a rapid linear increase in absorbance at 485 minus 575 nm occurred. The activity was not dependent on the addition of a mitochondrial respiratory substrate. It was further found that a corresponding oxygen consumption occurred when epinephrine was added to the aerobic potato mitochondria or submitochondrial particles. This basal rate of epinephrine oxidation was not found to such an extent in mung bean or tulip bulb mitochondria. The tyrosinase activity of the mitochondria was assayed with both pyrogallol and L-tyrosine as substrates. It was found that potato mitochondria possessed a significant level of activity (490 nmol of 02 consumed/mg/min at 25°C with 0.1 M pyrogall01 as substrate) compared to either tulip bulb or mung bean mitochondria (t15 nmol of O2 consumed/mg/min at 25°C with 0.1 M pyrogallol), which also did not show the rapid epinephrine oxidation capacity. Further, when epinephrine was added to a commercially purchased sample of partially purified mushroom tyrosinase, a rapid oxygen consumption ensued, forming a product which was spectrally identical to adrenochrome. The potato epinephrine oxidase activity was greater than 98% inhibited by both 1 mM KCN and 1 mM salicylhydroxamic acid, but was unaffected by 1 pg/ml of antimycin A (Fig. 1). It is already known that both KCN (15) and salicylhydroxamic acid (16) potently inhibit tyrosinase reactions. Furthermore, superoxide dismutase (1.5 pg/ml) inhibited the rate by less than 15%, an indication that free superoxide anions are not involved in the process. It is suggested that this activity of potato mitochondria is caused by a tyrosinase activity which is associated with them. The activity is probably a contaminant, since it was found that gentle osmotic shock treatment of the mitochondria (for example, injection into 200 mosM sucrose) caused much of the activity to be dislodged from

RICH

AND

BONNER

at

485m

575nm

%A?

\\

FIG. 1. Tyrosinase-mediated epinephrine oxidation by potato submitochondrial particles. Potato submitochondrial particles were resuspended at 0.13 mg of protein/ml in a medium containing 30 mM Tris-Mops at pH 8.0. The cuvette was placed in a double-beam spectrophotometer with wavelengths set at 485 minus 575 nm. The reaction was initiated by the addition of 1 mM epinephrine. Subsequent additions of 1 mM KCN, 1 InM salicylhydroxamic acid, or 1 pg/ml of antimycin A were made as indicated. Downward deflection indicates epinephrine oxidation.

the mitochondrial fraction, even though the mitochondrial outer and inner membranes were unaffected by the treatment [cf. Ref. (9)]. Furthermore, the activity associated with the mitochondria represents less than 2% of the total tissue tyrosinase activity, and the activity appeared to some extent in all subcellular fractions. Submitochondrial particles prepared from the potato mitochondria had significantly less tyrosinase activity (generally around 100-150 nmol of 02 consumed/ mg/min at 25°C with 0.1 M pyrogallol), but even this remaining level caused interference in the superoxide anion assays. Substrate-Dependent Superoxide Anion Generation by Cyanide-Sensitive Potato Tuber Submitochondrial Particles In order to eliminate the tyrosinase interference in potato mitochondrial or submitochondrial superoxide anion assays, it was necessary to perform the assays in the presence of KCN or salicylhydroxamic acid so that the tyrosinase was inhibited. Such an experiment performed with potato submitochondrial particles is illustrated in Fig. 2. The addition of NADH to salicylhydroxamic acid-inhibited submitochondrial particles caused on oxidation of epinephrine to adrenochrome (Fig. 2A). This activity was unaffected or slightly stimulated by anti-

FIG. 2. Superoxide anion generation by potato submitochondrial particles. Submitochondrial particles of potato tuber were resuspended to a protein concentration of 0.27 mg/ml in 30 mM Tris-Mops at pH 8.0. The appropriate initial inhibitor and 1 mM epinephrine were added and the oxidation of epinephrine was monitored at 485 minus 575 nm. Additions were made as indicated, and concentrations were: salicylhydroxamic acid, 1 mM; KCN, 1 mM; antimycin A, 1 pg/ml; superoxide dismutase, 1.5 pg/ml; NADH, 1 mM; succinate, 10 mM; ATP, 0.3 mM.

mycin A and was slightly inhibited by KCN (Fig. 2C). The identification of a role of superoxide anions in this process was shown by the sensitivity of the rate to superoxide dismutase (Fig. 2A). A further experiment was performed in the presence of 1 mM KCN. The addition of NADH promoted a rate of epinephrine oxidation which was slightly less than that in the presence of salicylhydroxamic acid. The subsequent addition of antimycin A caused little or no stimulation of the rate. Salicylhydroxamic acid, 1 mM, had virtually no effect on this rate (Fig. 2B). The superoxide dismutase sensitivity of this rate could not be tested, since cyanide inhibited the added dismutase (the only commeritally available superoxide dismutase that we could obtain was the iron-containing, i.e., cyanide-sensitive, type). When succinate was used as substrate, a little superoxide anion generation could be observed, and this was slightly stimulated by antimycin A. ATP was added in these assays to ensure maximum activation of the succinate dehydrogenase, but its absence did not lower the maximal rates obtained. The rate was inhibited by superoxide dismutase (Fig. 2D) and by 1 mM KCN (Fig. 2E).

SUPEROXIDE TABLE

GENERATION

I

EPINEIJHHINE OXIDATION AND OXYGEN CONSUMPTION RATES BY POTATO SIJBMITOCHONDRIAI. PARTICLES”

Conditions

Ianomoles per milligram per minute of 25°C pinephrim oxidation

Basal rates (+ epinephrine) 1.6 Control 0.5 +KCN 0.45 +Salicylhydroxamic acid 1.6 +Antimycin A 9.6 +Superoxide dismutase NADH-supported rates 1.6 (1.7 NADH + KCN (+ antimycin A) 1.5 (2.1 NADH + salicylhydroxamic acid (+ antimytin A) 1.45 NADH + salicylhydroxamic acid + antimycin A+KCN 0.2 NADH + salicylhydroxamic acid + antimycin A + superoxide dismutase iot tested NADH (+ cytochrome 4 Succinate-supported rates 0.9 Succinate + salicylhydroxamic acid 1.1 Succinate + salicylhydroxamic acid + antimycin A 0 Succinate + salicylhy. droxamic acid + anti mycin A + KCN 0.2 Succinate + salicylhy droxamic acid + anti. mycin A + superoxide dismutase Succinate (+ cyto. Vat tested chrome c)

T ?( c

)xygen consumption

7.7 <2 t2 7.7 Not tested <5 (.<5) 256 :5)

t5

1Not tested

256 (512)

154 <2

<2

Not tested

154 (307)

n Rates of epinephrine oxidation and oxygen consumption were measured (see Materials and Methods) and concurrently with the same preparation of particles. The results given are of a typical single experiment selected from at least five determinations. The measured tyrosinase rates, and hence basal rates, were rather variable between samples (range, 6-24 nmol of epinephrine oxidized/mg/min at 25”(Z), although the calculated superoxide anion generation rates (measured minus basal rate) were consistent (within 25% of the values obtained in this experiment). The low

1N PLANT

MITOCHONDRIA

2109

We were unable to detect significant endogenous superoxide dismutase activity in the submitochondrial particle preparations, which indicated that the rates measured were not diminished by rapid superoxide removal. A comparison of the results of a typical experiment on measurements of the rates of epinephrine oxidation and oxygen consumption in potato submitochondrial particles is presented in Table I. The basal rates of epinephrine oxidation and oxygen consumption in the absence of substrate (caused by tyrosinase) were sensitive to both KCN and salicylhydroxamic acid, but were insensitive to antimycin A and superoxide dismutase. When NADH was added in the presence of either KCN of salicylhydroxamic acid, an increase in the epinephrine oxidation rate over the basal rate (i.e., the superoxide anion generation rate) of about 1 nmol/mg/min was found, which could be slightly stimulated by antimycin A and potently inhibited by superoxide dismutase. The oxygen consumption rates were too low to measure under these conditions, since the substrate oxidation rate was highly sensitive to either KCN or antimycin A. Similar rates were observed when succinate was substrate, although the maximal superoxide generation rate (observed epinephrine oxidation rate minus basal epinephrine oxidation rate) was about 0.5 nmol/mg/min. The quality of the submitochondrial particle preparations was demonstrated by the fact that they could oxidize NADH and succinate at rates of oxygen consumption of 512 and 307 nmol of OX consumed/mg/min, respectively, in the presence of cytochrome c. Rates of Superoxide Anion Generation in Mitochondria which Possess an Alternative Oxidase Similar experiments were performed with mung bean mitochondria. The mitochondria had rates of oxygen consumption oxygen consumption rates are given as a maximum possible value, since it is not possible to measure such low rates accurately with standard oxygen electrode equipment. The figures in parentheses refer to rates after the further addition of the respective reagent (also given in parentheses).

210

RICH

AND

via the alternative oxidase of up to 30 nmol of O2 consumed/mg/min at 25°C and we were able to prepare submitochondrial particles with rates of 5-15 nmol of 02 consumed/mg/min at 25°C via the alternative pathway. Hence, it was possible to assay the rates of superoxide anion generation during the operation of the alternative pathway. Table II summarizes the salient features of the results which we obtained with mung bean submitochondrial particles. The control rates of epinephrine oxidation indicate a small but significant amount of tyrosine contamination of these particles. Hence, again the experiments were generally performed with the prior addition of either KCN or salicylhydroxamic acid to reduce this interference. The addition of NADH to the particles in the presence of salicylhydroxamic acid caused an increase of 5-10 nmol of epinephrine oxidized/mg/min in the basal epinephrine oxidation rate. This rate was generally TABLE EPINEPHHINE

II

OXIDATION

CONSUMPTION RATES SUBMITOCHONDHIAL

Conditions

AND OXYGF,N RY MUNG BEAN PAHTICLES”

Nanomoles per milligram per minute at 25°C Epinephrine oxidation

Control (+ epinephrine) +KCN +Salicylhydroxamic acid +Antimycin A NADH + KCN (+ antimycin A) NADH + salicylhydroxamic acid (+ antimycin A) NADH + KCN + antimytin A + salicylhydroxamic acid Succinate + KCN (+ antimycin A) Succinate + salicylhydroxamic acid (+ antimycin A) Succinate + KCN + antimycin A + salicylhydroxamic acid n See Table I, footnote

7.1 SC2 ~2 7.1 5.5 10.3 (12.8)

4.9

~2 (<2)

a.

Oxygen consumption Not tested Not tested Not tested Not tested 6.5 (6.5) 167 (12)

<2

7.4

~2 (2.7)

21

~2

<2

BONNER

slightly increased by antimycin A addition, but was completely inhibited by superoxide dismutase. KCN had a strong inhibitory effect on the NADH-supported rate, reducing it almost to the basal rate observed in the presence of salicylhydroxamic acid alone. When a similar experiment was performed with KCN present as the initial inhibitor, only a low rate of NADH-supported epinephrine oxidation was obtained, l-2 nmol/mg/min. The rate was virtually unaffected by antimycin A and salicylhydroxamic acid. When succinate was the respiratory substrate, the low substrate-induced rate (1-2 nmol of epinephrine oxidized/mg/min) was only discernible above the basal rate in the presence of antimycin A, and was totally inhibited by KCN (Table II). This result is similar to that obtained with the potato system. The alternative oxidase pathway operates maximally in the presence of substrate and either KCN, antimycin A, or both. In the case of mung bean submitochondrial particles, it can be deduced that oxygen consumption rates via the alternative pathway of about 6 and 7 nmol of 02 consumed/mg/min can occur with NADH and succinate as respiratory substrates, respectively (as judged by the KCN- and antimycin A-insensitive and salicylhydroxamic acid-sensitive oxygen consumption; see Table II). However, the rate of KCN- and antimycin A-insensitive and salicylhydroxamic acid-sensitive superoxide anion generation is less than 1 nmol of Oz/mg/min with either NADH or succinate as substrate. It is therefore concluded that the alternative oxidase does not produce free superoxide anions to a significant extent. This conclusion was demonstrated even more clearly when submitochondrial particles of Symplocarpus foetidus or Arum maculatum spadices were used, since it was easy to prepare submitochondrial particles from these tissues which retained an extremely active alternative oxidase. For example, the rate of KCN and antimycin Ainsensitive oxygen consumption by Symplocarpus foetidus submitochondrial particles with NADH as substrate was 129 nmol of

SUPEROXIDE

GENERATION

O2 consumed/mg/min, and yet superoxide anion generation was less than 1 nmol/mg/min under the same conditions (Table III). Similarly, the rate of antimycin A-insensitive (and KCN-insensitive) oxygen consumption in Arum maculatum submitochondrial particles was 556 and 180 nmol of 0, consumed/mg/min with NADH and succinate as substrates, and yet the superoxide anion generation rate was 50fold less than this. When whole mitochondria of Arum maculatum were used for a similar experiment, even lower rates of superoxide anion generation were observed (Table III), although this may have been caused in part by enzymes such as superoxide dismutase, which are present in mitochondria but largely absent from submitochondrial particles. DISCUSSION

From the preceding results, it is apparent that several pathways of oxygen consumpTABLE III EPINIWHRINE OXIDATION AND OXYGEN C~NSIJMPTI~N RATES BY Symplocarpus foetidus AND Arum maculatum MITOCHONDRIA AND SIJRMITOCHONDRIAI, PAHTICLES” Tissue and conditions

Nanomoles per milligram per minute at 25OC Epinephrine oxida tion

Symplocarpus foetidus Control (+ epinephrine) NADH NADH + KCN + antimycin A Arum maculatum Submitochondrial particles Control (+ epinephrine) NADH + antimycin A Succinate + antimycin A Whole mitochondria Control (+ epinephrine) NADH + antimycin A Succinate + antimycin A ” See Table I, footnote

a.

Oxygen consumption

3.4 8.0 1

Not tested 141 129

2.6

Not tested

9.5 3.1

556 180

0.3

-

2.0 0.7

758 368

IN PLANT

MITOCHONDRIA

211

tion are potentially available to isolated plant mitochondria or submitochondrial particles. These can be identified as follows: (A) Oxygen consumption via cytochrome oxidase to produce water, a process which accounts for greater than 95% of the oxygen consumption in normal, cyanide-sensitive mitochondria. (B) Oxygen consumption via the alternative oxidase to produce eventually water, perhaps via hydrogen peroxide (B), but not involving superoxide anions. The process is unaffected by cyanide and antimycin A, but is inhibited by hydroxamic acids (17). (C) Direct reduction of oxygen to superoT:ide anions in the flavoprotein region of the NADH dehydrogenase segment of the respiratory chain. The component responsible is likely to be the flavoprotein (of either internal or external dehydrogenase) or perhaps an iron sulfur center. The process may be identified by its insensitivity to KCN, antimycin A, and salicylhydroxamic acid and by the sensitivity of the assayed epinephrine oxidation rate to superoxide dismutase. (D) Oxygen reduction to superoxide anions in the ubiquinone-cytochromes b region of the respiratory chain. The process may be identified by its insensitivity to salicylhydroxamic acid and antimycin A, its sensitivity to KCN, and the sensitivity of the assayed rate to superoxide dismutase. In this latter case, the properties are analogous to those of superoxide anion generation in mammalian systems [cf. Refs. (3-6)]. The property of antimycin A insensitivity and KCN sensitivity allows us to distinguish this process from that occurring at the flavoprotein level (process C). The cyanide sensitivity is difficult to rationalize in classical linear schemes of electron transport, but may be explicable in a protonmotive ubiquinone cycle type of arrangement, as proposed by Mitchell [cv. Refs. (18, 19)]. In these schemes, fully reduced ubiquinone donates an electron to cytochrome cl, and leaves an unstable, highly reducing semiquinone species which would normally reduce cytochrome bsG6.Antimytin A would tend to increase, and KCN would prevent, the production of the semiquinone species. It is presumably this un-

212

RICH

AND

stable semiquinone or a closely interacting species which reduces the oxygen to superoxide anion, since only species at this site would have enough reducing potential for the reaction [the oxygen/superoxide couple has an E, at pH 7 of around -330 mV (20)]. (E) Oxygen consumption (and epinephrine oxidation) by a contaminating tyrosinase enzyme, when a suitable substrate is added. The process is easily identifiable, since it is not dependent upon the addition of respiratory substrate, is insensitive to superoxide dismutase and antimycin A, and is potently inhibited by both KCN and salicylhydroxamic acid. The products of the reaction are oxidized substrate and water (15, 21). These possible pathways are illustrated in Fig. 3. It should be noted that by far the most important oxygen-consuming pathways are those of the alternative and cytochrome oxidases. The relative activities of these have already been well documented in many systems [for example, see reviews in Refs. (7, 22)]. The two pathways of superoxide anion generation are not implied to be physiologically significant. Instead, it is thought that they represent a nonphysiological “short-circuiting” of the electron transport chain. Reactions of molecular oxygen with flavoproteins, quinones, and iron sulfur centers are well-known chemical events (23-25). An idea of the relative con-

FIG. 3. A summary of the routes of oxygen reduction by a higher plant mitochondrial fraction. Fp, flavoprotein, FeS, iron sulfur centers. The tyrosinase route included here is not intended to indicate that the reaction is an integral part of mitochondrial function. Instead, it is probably a contaminant which is present to some extent in most mitochondrial preparations (see text).

BONNER

tribution of the pathways C and D to the total superoxide anion generation rate may be gained by testing the effect of KCN on hydroxamic acid-inhibited mitochondria in the presence of NADH and antimycin A (cf. Fig. 2C). The percentage inhibition of the rate by KCN represents the percentage of the rate provided by pathway D (the ubiquinone-cytochrome b region), since only this part is cyanide sensitive. Hence, for example, in submitochondrial particles of potato with NADH, about lo-30% of the rate is via process D, and with succinate, 100% of the rate is via process D. The pathway involving tyrosinase is presumably not related to mitochondrial function. It is likely that it either is present in a contaminating organelle or has become attached to the mitochondrial membrane during the isolation procedure. It is emphasized here merely to point out an often large and misleading interfering factor in the determination of superoxide anion generation rates in plant mitochondria. We were able to observe an antimycin Ainsensitive hydrogen peroxide production in mung bean mitochondria and submitochondrial particles (8). In submitochondrial particles, the rate was sufficient to account for the antimycin A-insensitive oxygen consumption rate, and hence we suggested the possibility of hydrogen peroxide as a major product of the alternative oxidase. We were unable to test the effects of cyanide and hydroxamic acids, since these interfered with the cytochrome c peroxidase assay. The results of the work described in this manuscript, however, suggest that much of the hydrogen peroxide generation observed in this system may be via the “nonphysiological” alternative pathway of oxygen reduction to superoxide by the flavoprotein and semiquinone components (and hence to peroxide via dismutation). This pathway may represent a large proportion of the cyanide- and antimycin A-insensitive oxygen consumption in some preparations. The question still remains to be established, however, whether it is possible to “trap” a peroxide intermediate during alternative oxygen consumption. Some recent data from this laboratory suggest that this may be feasible. For example, the near-doubling

SUPEROXIDE

GENERATION

of the alternative oxygen consumption rate on the addition of bovine serum albumin in mung bean mitochondria (P. R. Rich, unpublished data) is difficult to rationalize without postulating a peroxide intermediate which may be stablized or trapped in some way by the bovine serum albumin. ACKNOWLEDGMENTS The authors wish to thank Ms. N. K. Wiegand for expert technical assistance and Ms. M. Mosley for preparation of the manuscript. This work was supported by grants from the National Science Foundation and the Herman Frasch Foundation. REFERENCES 1. CI.ARK, W. M. (1960) in Oxidation-Reduction Potentials of Organic Systems, p. 25, Waverly Press, Baltimore. 2. BOVEHIS, A., AND CHANCE, B. (1973) Biochem. J. 134, 707-716. 3. LOSCHEN, G., FLOHIX, L., AND CHANCE, B. (1972) FEBS Lett. 18, 261-264. 4. BOVERIS, A., OSHINO, N., AND CHANCE, B. (1972) Biochem. J. 128,617-630. 5. BOV~RIS, A., AND CADENAS, E. (1975) FEBS Lett. 54.311-314. 6. CADF,NAS, E., BOVEIIIS, A., RAGAN, C. I., ANI) STOPPANI, A. 0. M. (1977) Arch. Biochem. Biophys. 180, 248-257. 7. HENIIY, M-F., ANI) NYNS, E-J. (1975) Sub-Cell. Biochem. 4, l-65. 8. RICH, P. R., BOVEHIS, A., BONNE:H, W. D., JH., AND MOOHE, A. L. (1976) Biochem. Biophys. Res. Commun. 71,695-703. 9. DOLJ~E, R., MANNF.I.LA, C. A., AND BONNER, W.

IN PLANT

10.

11. 12. 13. 14.

15. 16. 17.

18. 19. 20. 21.

22. 23. 24. 25.

MITOCHONDRIA

213

D., JH. (1973) Biochim. Biophys. Acta 292, 105-116. BONNER, W. D., JH. (1967) in Methods in Enzymology, Vol. X, pp. 126-133, Academic Press, New York. RICH, P. R., AND BONNER, W. D., JIM. (1978) Biochim. Biophys. Acta, 501,381-395. LOSCHEN, G., Azz~, A., AND FLOHE, L. (1973) FEBS Lett. 33, 84-88. GHERN, S., MAZUR, A., AND SHOHII, E. (1972) J. Biol. Chem. 220, 237-255. LOWRY, 0. H., ROSEBHOCJGH, N. J., FAHH, A. L., AND RANDALI., R. J. (1951) J. Biol. Chem. 193, 265-275. MASON, H. S. (1965) Annu. Reu. Biochem. 34, 595-634. RICEI, P. R., AND BONNF.R, W. D., JH. (1977) Plant Physiol. 59, 60P. SCHONBAUM, G. R., BONNE:H, W. D., JH., STOHEY, B. T., ANDBAHH, J. T. (1971) Plant Physiol. 47, 124-128. MITCHF,LL, P. (1976) J. Theor. Biol. 62, 327-367. RICH, P. R., AND Moort~, A. L. (1976) FEBS Lett. 65,339-344. WOOD, P. M. (1974) FEBS Lett. 44, 22-24. MAKINO, N., MCMA~IILL, P., MASON, H. S., ANr> Moss, T. H. (1974) J. Bio. Chem. 249, 6062-6066. IKIJMA, H. (1972) Annu. Reu. Plant Physiol. 44, 126-134. FRIDOVICH, I. (1974) A&an. Enzymol. 41, 36-97. MISKA, H. P., AND FRIDOVICH, I. (1972) J. Biol. Chem. 247, 188-192. NAKAMURA, T., YOSHIMIJHA, J., NAKAMIJHA, S., ANI) O~IJI