Superoxide and hydrogen peroxide production in cyanide resistant Arum maculatum mitochondria

Plant Science Letters, 11 (1978) 351--358

351

© Elsevier/North-Holland Scientific Publishers Ltd.

SUPEROXIDE AND HYDROGEN PEROXIDE PRODUCTION IN CYANIDE RESISTANT A R UM MA C ULA T U M MITOCHONDRIA

S. H U Q and J.M. P A L M E R Department of Botany, Imperial College, University of London, Prince Consort Road, London S W 7 2BB (Great Britain) (Received September 23rd, 1977) (Accepted December 14th, 1977)

SUMMARY

Submitochondrial particles from A r u m maculatum containing a powerful cyanide insensitive oxidase were assayed by various methods to determine the end product of its interaction with oxygen. Using cytochrome c peroxidase to assay the production of H202 it was possible to detect H202 formation by A r u m submitochondrial particles oxidizing NADH but not when oxidizing succinate. The rate of production of H~O2, however, was insufficient to account for the rate of oxygen uptake due to the alternate oxidase. The production of superoxide was determined using the luminol and adrenochrome assays. It was found that some superoxide was produced when A r u m submitochondrial particles oxidized NADH but not when they oxidized succinate and again at insufficient rates to account for the rate of oxygen uptake by the alternate oxidase, stoicl~iometric determination of the ratio of NADH oxidized to oxygen taken up in the presence of 1 mM KCN, sufficient to inhibit catalase activity such that added peroxide remains stable, showed H20 to be the only detectable product. It is suggested that although both H20~ and superoxide are produced by A. maculatum submitochondrial particles this is not due to the alternate oxidase but may be due to another component of the respiratory chain possibly at the level of the NADH dehydrogenase.

INTRODUCTION

It has generally been assumed that water was the product formed when the Address correspondence to: S. Huq, Department of Botany, Imperial College, Prince Consort Road, London SW7 2BB, England. Abbreviations: CLAM, m-chlorobenzohydroxamic acid; FCCP, carbonyl cyanide, p-trifluoromethoxyphenylhydrazone; HRP, horseradish peroxidase; SHAM, salicylhydroxamic acid.

352 cyanide insensitive oxidase, present in some plant mitochondria, reduces oxygen. Recent data from Rich et al. [1] show that hydrogen peroxide is produced in mung bean mitochondria which contain a cyanide insensitive respiratory pathway. We have looked at mitochondria isolated from A r u m maculatum which contain a powerful cyanide insensitive oxidase. Since little is known about the alternative oxidase itself it would be useful to identify the nature of the end product of its interaction with oxygen. This would indicate whether the interaction was a one-electron process, in which case the expected product would be superoxide, a two-electron process, in which case the expected product would be hydrogen peroxide, or a four-electron process, in which case the expected product would be water. We have used several different assays to detect the formation of hydrogen peroxide: (a) using cytochrome c peroxidase [2] ; (b) by calculating the stoichiometry between the a m o u n t of oxygen consumed and NADH oxidized; (c) by determining the effect of horseradish peroxidase (HRP), which would stabilize any H202 produced, on the rate of oxygen uptake by Arum submitochondrial particles, in the presence of cyanide [1]. The superoxide radical was detected by (a) measuring the chemiluminescence, produced by the interaction of the superoxide radical with luminol [3] ; (b) following the formation of adrenochrome when epinephrine interacts with superoxide [ 3].

EXPERIMENTAL Mitochondria and submitochondrial particleswere prepared from A. maculatum spadices by the method of C a m m a c k and Palmer [4]. The submitochondrial particleswere stored at 77 K. Oxygen uptake was measured polarographicaUy using a Clark-type oxygen electrode. The reaction medium used was 0.3 M sucrose, 10 m M N-[trishydroxymethyl)methyl]-2-aminoethanesulphonic acid, 5 m M KI-12PO4 and 5 m M MgCl2 at p H 7.2. Protein was assayed by the method of Lowry et al. [5] aftersolubilization with 0.2 ml (10% w/v) deoxycholate in a finalvolume of 5.9 ml. Cytochrome c peroxidase was prepared from bakers' yeast by the method of Yonetani and Ray [2]. H R P was obtained from the Boehringer Corporation Ltd, epinephrine and xanthine from Sigma Chemical Co. Ltd, salicylhydroxamic acid (SHAM) from Aldrich Chemical Co. Ltd, and luminol (5-amino-2,3dihyclro-l,4-phthalazinedione) and xanthine oxidase from B D H Chemicals Ltd. m-Chlorobenzohydroxarnic acid (CLAM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were generous gifts from Professor J.L. Harley and Dr P.G. Heytler respectively.Superoxide dismutase was a generous gift from L. Henry. Antimycin A was obtained from Calbiochem Ltd. RESULTS Using the cytochrome c peroxidase assay, Rich et al. [1] found that in intact

353

m u n g bean mitochondria oxidizing either succinate or NADH, in the presence of antimycin A, H202 production accounted for less than 5% of the observed oxygen consumption. Using m u n g bean submitochondrial particles t h e y f o u n d H202 production able to account for the observed antimycin A-insensitive oxygen c o n s u m p t i o n with NADH as the substrate; t h e y reported that the submitochondrial particles were unable to oxidize succinate adequately as a substrate. A. maculaturn submitochondrial particles easily oxidized succinate at rates of between 400--1000 nmol • rain -~ • mg-~ protein and NADH at over 600 nmol • min-~ • m g -1 protein in the presence o f antimycin A compared with less than 20 n m o l • min -~ • rag-~ protein for mung bean submitochondriai particles [ 1 ]. Using the c y t o c h r o m e c peroxidase assay [2] (Fig. 1) there is evidence of H202 formation with A r u m submitochondrial particles oxidizing NADH followed by a fairly rapid breakdown of the e n z y m e substrate complex upon anaerobiosis. With succinate, however, there is no evidence of H202 formation

I

I

I

i

I

'

I I I

I

I=AoD 0.001

II I I I

! I t o.o5 mM NADH

" I ' 10 m M succinate

H202 !

I

1 rain

Fig. 1. Formation of cytocbxome c peroxidase --H~O2 complex by A r u m submitochondrial particles oxidizing NADH or succinate in t h e p r e s e n c e of antimycin A. Measured in an Aminco DW2 dual beam spectrophotometer with reference at 407 nm, sample at 419 nm, band pass 2 nm. 1 ml potassium phosphate buffer (0.1 M, pH 7.0) contained A r u m submitochondrial particles (0.35 mg protein), 500 ng antimycin A, 2 X 10 -7 M FCCP and 20/~1 cytochrome c peroxidase.

354 0.2raM NADH

nmol oxygen consumed p e r 0.2 m M N A D H

0.04 mM NADH 0. O8 m M N A D H

•

l

NADtt

I 1 min

Fig. 2. Stoichiometric determination of product of NADH oxidation in A r u m submitochon~rial particles in the presence of KCN. Reaction medium (1 ml) contained submitochondrial particles (0.35 mg protein), 2 × 10 -~ M FCCP and 1 mM KCN. Average ratio of oxygen used to NADH consumed = (37 nmol NADH/19.13 nmol 02) --- (2 nmol NADH/ 1 nmol 02). This gives an equivalence of 1 mol of oxygen to 2 tool of NADH ffi H20. a l t h o u g h a d d e d H202 c o u l d be d e t e c t e d . T h e initial rate o f H202 p r o d u c e d was a b o u t 30 n m o l • m i n -1 • m g -1 p r o t e i n using AemM ( 4 1 9 - - 4 0 7 n m ) = 5 0 1 • mmo1-1 • c m -1 [2] w h i c h is a n o r d e r o f m a g n i t u d e t o o small t o a c c o u n t f o r t h e o x y g e n u p t a k e o f t h e a l t e r n a t i v e p a t h w a y . This figure f o r H202 prod u c e d has b e e n c o r r e c t e d t o a c c c o u n t f o r a c o n t a m i n a n t in t h e c y t o c h r o m e c p e r o x i d a s e p r e p a r a t i o n o f an N A D H o x i d a s e c a p a b l e o f p r o d u c i n g s o m e H202 in t h e a b s e n c e o f a n y s u b m i t o c h o n d r i a l particles. A r u m s u b m i t o c h o n d r i a l particles were f o u n d t o have catalase a c t i v i t y as TABLE I EFFECT OF ADDED HRP 10 ~/mg MITOCHONDRIAL PROTEIN ON RATE OF OXIDATION OF NADH AND SUCCINATE BY A r u m SUBMITOCHONDRIAL PARTICLES IN THE PRESENCE OF KCN AND KCN + CLAM Substrate

Rate of oxidation nmol 02 • min -1 • mg-* protein + Antimycin A

NADH Succinate

+ Antimycin A + CLAM

-HRP

+HRP

-HRP

+HRP

570 400

500 440

17 --

60 --

355

measured by oxygen evolution from added peroxide. This catalase activity was fully inhibited by 1 mM KCN. The experiments to measure the stoichiometry of o x y g e n c o n s u m e d and N A D H oxidized were all carried out in the presence of I mM KCN. The a m o u n t of oxygen c o n s u m e d by the oxidation of a given a m o u n t of N A D H can be calculated from the oxygen electrode trace as shown in Fig. 2. The concentration of N A D H was determined enzymaticaUy and the concentration of oxygen was taken as 240 n m o l / m l at 25°C. The ratio of oxygen consumed in the oxygen electrode to N A D H oxidized was thus calculated and found to be 19.1 nmol of 02 : 37 nmol of NADH. Hence the stoichiometrically detectable product is water. Rich et al. [1] reported a doubling of the rate of oxidation via the alternative oxidase in mung bean mitochondria in the presence of added HRP to "trap" the H202 produced. Table I shows the effect of added HRP on the rate of oxidation of N A D H and succinate in the presence of antimycin A by Arum 800

/

600 cN

c;

400 > 0

0 o

r~ 0 E~

200

0

I

~

20

40

o

o

60

80

I

i

I

I00

~i AP~m submitochondrial particles

Fig. 3. Initial chemiluminescence of luminol in the presence of different concentrations of Arum submitochondrial particles oxidizing NADH (o) and succinate (o) as substrates. A ~ a y

mixture contained 3 ml tricine buffer (5 mM, pH 8.5), submitochondrial particles (50 mg protein/ml), 25 mM NADH and 20 mM succinate.

356 submitochondrial particles. This shows only a 10% increase in the rate of oxidation of succinate and an actual decrease in the rate of oxidation of NADH. Table I also shows the existence of an antimycin A and CLAM insensitive oxidation which is of the same order of magnitude as the antimycin A ip~sensitive p a t h w a y in mung bean [1], and is almost quadrupled in the presence of HRP. The superoxide radical can be detected by the chemiluminescence produced 5 ~i superoxide

dismutase

&on = o.oi

,

5 mM s u c c i n a t e

C

l

Fig. 4. Superoxide production by Arum submitochondrial particles. Superoxide production detected by adrenoehrome formation measured in dual beam spectrophotometer with reference at 575 nm, sample at .480 rim, ban d pass 2 rim. Assay conditions: (A) 0.8 ml tricine buffer (5 mM, pH 8.5) conizlnln_gsubmitochondrial particles (0.8 mg protein) + 2 X 10 -~ M FCCP + 1 mM e p ~

mglml) 0.8 EUImg p ¢ ~ , submitochondrial ~

+ 0.2 ml saturated xanthtne, xanthine oxids~ (5

supm~xide dismutme. (B and C) 1 ml ttddne buffet containing (0.8 lag ~ ) + 2 x 10" M ~ + 1 mM epinephrine, sub-

strate concentrations as indleated.

357 when it interacts with luminol [3]. This was measured in counts per minute on the tritium channel of a Packard Tri-Carb liquid scintillation spectrometer model C2425. However, this method is a qualitative rather than quantitative measure of superoxide production. A r u m submitochondrial particles showed an initial burst of chemiluminescence when oxidizing NADH in the presence of luminol. The intensity of the initial burst of chemiluminescence depended on the concentration of submitochondrial particles (Fig. 3). No luminescence was produced when succinate was oxidized. The initial burst of chemiluminescence by A rum submitochondrial particles oxidizing NADH was seen to be suppressed by superoxide dismutase and CLAM as well as by KCN and ethanol. Adrenochrome is formed by the reaction of the superoxide radicals on epinephrine [3]. Fig. 4 shows the formation of adrenochrome in the presence of Arurn submitoch0ndrial particles when xanthine reacts with xanthine oxidase to produce superoxide which is suppressed by added superoxide dismutase. When A r u m submitochondrial particles oxidized NADH there was also some adrenochrome formation which began to decrease after about 1.5 rain. A r u m submitochondrial particles oxidizing succinate, however, showed no adrenochrome formation at all. The rate of adrenochrome formation by A r u m submitochondrial particles oxidizing NADH was 8.3 nmol • rain-' • mg-1 protein of adrenochrome formed, using e48s-sTs of 2.96 m M - 1 • cm-' [6], which can account for only a small fraction of the oxygen uptake by these submitochondrial particles.

DISCUSSION The results suggest that although hydrogen peroxide and superoxide are produced by A r u m submitochondrial particles,they are only produced when the preparation oxidizes N A D H and not when it oxidizes succinate.Neither of these products is produced at a rate sufficientto account for the oxygen uptake by the alternativeoxidase. Furthermore, studies of the stoichiometry gave a ratio of oxygen consumed to N A D H oxidized indicatingwater to be the product. The rate of oxidation of N A D H and succinate via the cyanide insensitive pathway, in A. maculatum submitochondrial particlesdid not double in the presence of H R P which would be expected to "trap" any H202 produced as an enzyme substratecomplex, as reported with m u n g bean submitochondrial particles[1]. However, in A. maculatum there is a considerable rate of oxygen uptake which is insensitiveto both K C N and C L A M , and is of the same order of magnitude reported for the alternativepathway in mung bean [1]. This residualrate is capable of being stimulated by the presence of HRP. It m a y therefore be possible that the hydrogen peroxide is produced by the interactionof a respiratorychain with oxygen at a siteother than eitherthe cytochrome or alternative oxidase as has been reported in Ascaris lumbricoides "[7], pigeon heart mitochondria [8] and rat liver mitochondria [9]. It is known that flavoproteins can generate H202 [10]. It is therefore possible that the small amounts of H202 being produced by A r u m maculatum submitochondriai

358 particles m a y be due t o a flavoprotein, perhaps at t he N A D H dehydrogenase level r a t h e r t h a n f r o m any terminal oxidase. ACKNOWLEDGEMENTS This research was s u p p o r t e d by grants f r o m t he Science Research Council, T h e R o y al S o ciety and T h e Central Research Fund, University o f L o n d o n . S. H u q wishes t o acknowledge r e c e i pt o f a studentship f r o m t he Tropical P r o d u c t s Institute. REFERENCES 1 P.R. Rich, A. Boveris, W.D. Bonnet Jr. and A.L. Moore, Biochem. Biophys. Res. Commun., 71 (1976) 695. 2 T. Yonetani and G.S. Ray, J. Biol. Chem., 240 (1965) 4503. 3 G. Loschen, A. Azzi, C. Richter and L. Floh~, FEBS Lett., 42 (1974) 68. 4 R. Cammack and J.M. Palmer, Biochem. J., 165 (1977) 347. 5 0 . H . Lowry, N.J. Rosehrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 6 S. Green, A. Mazur and E. Short, J. Biol. Chem., 220 (1956) 237. 7 E. Bueding and B. Charms, J. Biol. Chem., 196 (1952) 615. 8 G. Loschen, L. Flohd and B. Chance, FEBS Lett., 18 (1971) 261. 9 A. Boveris, N. Oshino and B. Chance, Biochem. J., 128 (1972) 617. 10 M. Dixon, Biochim. Biophys. Acta, 226 (1971) 269.