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
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
