Oxygen activation and Oxygen toxicity

Ann. Rev. Plant Physiol. 1982. 33:73-96 Copyright @ 1982 by Annual Reviews Inc. All rights reserved OXYGEN ACTIVATION AND OXYGEN TOXICITY Erich F. Elstner Technische Universitat Miinchen, Institut fUr Botanik und Mikrobioiogie, 8000 Miinchen 2, Arcisstrasse 21, West Gennany CONTENTS INTRODtTCTION ... .. .... . . ........ . . . . ....... . . . ... .. .. . .. . . .... . . . .. ... ....... .. . .. .. . . .... .. . .. .. . . ... . ........... . . . 74 OXYGEN ACTIVATION AND REACTIVE OXYGEN SPECIES ... ..... . . . . . ..... . .. ... 75 Reductive Activation .......... ........ . . ......... ... . . . ..... . ..... .. ....... . . . . .. ....... . . ............ . . .............. 75 Oxygen Addition to Organic Radicals or Reduced Metal Complexes .................... 75 Photodynamic Reactions ......................................... ,.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . 76 REACTIVITY AND DETECTION OF ACTIVE OXYGEN SPECIES ................ 77 Superoxide ........................................ . . . . . ... . . . . ....... . . . . . . . . ....... . . . . . . . . ..... ......... ........... ..... 77 Hydrogen Peroxide (H20JJ ........................................................................................ 77 The OR Radical ....................................................... .......................... ....... . . .............. 78 Singlet Oxygen .......................................................................................................... 78 Organic Peroxides and Peroxy Radicals ..................... .......... .......... ........... ......... . . . . . 79 OXYGEN ACTIVATION IN DIFFERENT COMPARTMENTS AND ORGANELLES OF PLANT CELLS ............................................ ............ 79 Peroxides in Cell Walls ........................... . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Microsomal Oxygen Activation .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Peroxisomes ............... .............. ........ . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . ..... .................... ...... 80 Oxygen Activation and Detoxification in Mitochondria .......... ............ ............. . ...... 80 Chloroplasts ....... .................. .... ...... .......... ..... .. . . . ..... . . . ...... . . . . . . . . ..... . . . . . ......... .......... . ... 81 Physiological observations and the biological significance of oxygen reduction ................ 8 1 Mechanisms o f oxygen activation in the chloroplast .... ............... ............. ............... ..... 82 Functions of activated oxygen species in chloroplasts ..... ............ . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 84 ENDOGENOUS MECHANISMS OF PROTECTION AGAINST DELETERIOUS OXYGEN SPECIES ....................... ...... ......................... 84 PATHOLOGICAL, ENVIRONMENTAL, AND COMMERCIAL ASPECTS OF OXYGEN TOXICITy ............................................................ 86 CONCLUDING REMARKS AND FUTURE TRENDS .................. ....... ............... 88 73 0066-4294/82/0601-0073$02.00 A nn u. R ev . P la nt . P hy sio l. 19 82 .3 3: 73 -9 6. D ow nl oa de d fro m w w w .an nu al re vi ew s.o rg by Z he jia ng U niv ers ity on 08 /11 /12 . F or pe rso na l u se on ly. 74 ELSTNER INTRODUCTION Of the gases present as major components of the atmosphere, dinitrogen (N2), dioxygen (02), and carbon dioxide (C02) are metabolized by living organisms. In the scientific literature we find the terms "nitrogen fixation" and "C02 fixation" but rarely "oxygen fixation." This is probably because both N2 and CO2 are incorporated into organic molecules while O2 is mainly utilized as a hydrogen acceptor yielding water. There are several biochemical pathways and reactions such as hydroxylations and oxygena­ tions, however, by which O2 is also incorporated into organic molecules. If we compare the biochemical mechanisms responsible for these fixations, we find that in the cases of N2 and COb only a few well-defined enzymic systems (nitrogenase, RuBP carboxylase, phosphoenolpyruvate carboxy­ lase) are operating, while oxygen incorporation can occur via widely differ­ ing mechanisms such as enzyme catalysis (RuBP -oxygenase, hydroxylases, phenoloxidases), chemical reactions (reduction of oxygen and incorporation of reduced species), and physical (photodynamic) activation. The complex situation encountered in the case of oxygen incorporation into organic molecules occurs because molecular oxygen in its ground state contains two unpaired electrons with parallel spins ("triplet" ground state) rendering dioxygen rather unreactive toward other molecules existing in the more common "singlet" ground state with paired electrons (95, 96). For the purposes of a reaction between triplet O2 and singlet molecules, the triplet state of oxygen has to be changed in order to circumvent this spin restric­ tion. In the course of these changes, several reactive oxygen species are gener­ ated. Thus, the respective oxygen-activating reactions have to be kept under strict control in the living cell in order to avoid detrimental effects. Under certain circumstances, these effects are deliberately introduced and utilized in competition between organisms and also by man, for example in medicine or in weed control. In the last decade, the field of oxygen physiology and biochemistry has increased worldwide, an advance undoubtedly triggered by a report in 1969 from McCord & Fridovich (130) on the superoxide dismutase activity of a well-known copper-protein, erythrocuprein. Since then, several books, proceedings of international conferences and reviews on oxygen metabolism, toxicity, and detoxification have appeared (14a, 15, 16,21, 32, 51, 95-97, 117, 135, 155, 165, 178, 194, 196). In this review, oxygen activation and toxicity in the plant cell will be discussed with special reference to the chloroplast. In several cases, microbial, animal, and medical aspects will be mentioned for the sake of clarification or compari­ son. A nn u. R ev . P la nt . P hy sio l. 19 82 .3 3: 73 -9 6. D ow nl oa de d fro m w w w .an nu al re vi ew s.o rg by Z he jia ng U niv ers ity on 08 /11 /12 . F or pe rso na l u se on ly. ACTIVATED OXYGEN 75 OXYGEN ACTIVATION AND REACTIVE OXYGEN SPECIES Reductive Activation During the successive univalent reduction of dioxygen, the species superox­ ide (02'-, HO ;), hydrogen peroxide (H202), hydroxyl radical (OH"), and finally water are formed according to the following scheme (89, 171, 176): (a) (b) (c) (d) This reaction chain requires initiation for the first endothermic step ( a) with Eo' for the redox couple O2 / 02'- of -0.33 V [equivalent to -0.16 V on a molar basis (reviewed in 63,64)], whereas the subsequent steps are exother­ mic and occur spontaneously, either catalyzed (c, b) or uncatalyzed (b, d) with rate constants of between ca 105 (b, uncatalyzed) and ca 1010 [diffusion controlled, reaction d (197)]. Various systems have been re­ ported as being able to act as electron donors in the above reaction chain and comprise components of biological electron transport systems (photo­ synthetic, mitochondrial, microsomal), substrates of autoxidizable enzymes (oxidases such as xanthine oxidase), autoxidizable organic molecules [such as 6-hydroxydopamine (34)], reduced metal ions (134), and solvated elec­ trons [produced by radiation or sonication (20, 21, 196)]. Through the catalysis of certain enzymes, two-electron reduction (yielding H202) and especially the four-electron reduction (producing water) are able to proceed without detectable radical intermediates and largely contribute to the oxy­ gen consumption of aerobic organisms. Oxygen Addition to Organic Radicals or Reduced Metal Complexes Several reactions have been described in which ground state dioxygen is incorporated into organic molecules. These reactions require either ac­ tivated receptor molecules such as organic radicals, R (131, 156), or metal complexes activating oxygen via "oxenoid" or related mechanisms [P 450, Udenfriend, Fenton, or Hamilton systems (89)]. The products of these processes are reactive peroxy radicals in the first case according to: R+02 -ROO' 2. and, in the latter case, epoxidized and / or hydroxylated products (96). As is evident from Equation 2, the activating step in this reaction is the produc­ tion of the acceptor radical, for example, by hydrogen abstraction. This can A nn u. R ev . P la nt . P hy sio l. 19 82 .3 3: 73 -9 6. D ow nl oa de d fro m w w w .an nu al re vi ew s.o rg by Z he jia ng U niv ers ity on 08 /11 /12 . F or pe rso na l u se on ly. 76 ELSTNER be accomplished enzymatically, as in the cases of the lipoxidase reaction with an unsaturated fatty acid (131, 191), peroxidase acting on indoleace­ tate (200), or by reaction of an organic molecule with other radicals, e.g. OR': RH + OR'-_. R + H20. 3. Peroxy radicals in turn can react further, initiating chain reactions and producing reactive alkoxy radicals after (metal-catalyzed) homolytic cleav­ age of the corresponding hydroperoxides: ROO' + RH -R + ROOH ROOH + Mn+ --_10 OH- + RO+ M (n+l) + Photodynamic Reactions 4. 5. Oxidations and oxygenations which are both dependent on molecular oxy­ gen and light have been known for many years and are of great chemical and biological importance (68, 69, 117). These photodynamic reactions appear to be caused by physical activation of ground state dioxygen by a "photosensitizing" light receptor pigment (P= pigment in the ground state; Ip, 3p = pigment in the singlet and triplet excited states, respectively), yielding either highly reactive singlet oxygen 102 (I �g; energy state = 22 kcal / mole) by direct energy transfer, according to p + h . v _1p_3p; 3p + 02--P + 102 (type 2, high efficiency) or by electron transfer reactions, after charge separation according to P + h· v_1p_3p; 3p + 02-P+' + 02'- (type 1, low efficiency) 6. 7. yielding an oxidant (p+) and superoxide, 02'- (68, 69). Several biological, photodynamic reactions and destructive processes involving 102 (reaction type 2) have been recently described (see below). A reasonable explanation for the observed damage may be due to the fact that 102 can yield hy­ droperoxides directly from unsaturated fatty acids (in membranes) accord­ ing to RH + 102 --_. ROOH, where ROOH can undergo 8. further reactions (see 4 and 5), finally resulting in visible signs of destruction (69, 131). A nn u. R ev . P la nt . P hy sio l. 19 82 .3 3: 73 -9 6. D ow nl oa de d fro m w w w .an nu al re vi ew s.o rg by Z he jia ng U niv ers ity on 08 /11 /12 . F or pe rso na l u se on ly. ACTIVATED OXYGEN 77 REACTIVITY AND DETECTION OF ACTIVE OXYGEN SPECIES Superoxide O2'- (or its corresponding acid, perhydroxyl radical HO ; ; pKa = 4.8) is a nucleophilic reactant with both oxidizing and reducing properties (63,64, 73, 74, 86, 171). With regard to molecules of biological importance, O2'- can oxidize sulfur compounds, o-diphenols, ascorbic acid (4, 9,,34, 53, 61, 136a, 137, 140), or NADPH (l8, 84), and has been shown to reduce cytochrome c' (128, 130), metal ions and metal complexes (87, 129, 187). Of particular importance is the dismutation of O2'- which results in the formation of H202, a reaction which occurs spontaneously with a rate strongly dependent upon the pH, or is accelerated by several orders of magnitude via catalysis by the enzyme superoxide dismutase [SOD, E.C. 1.15.1.1. (72)]' according to O2'- + 0z'- + 2H+ � Hz02 + O2 9. At physiological pH, reaction 9 proceeds with a rate constant of ca 105 M-I sec-I whereas the SOD-catalyzed (first order) rate proceeds under the same conditions with a rate constant of 2 X 109 M-I sec-I (63). Superoxide formation in biological systems may be detected by coupled reactions based on the reducing or oxidizing activities of O2'-, where the indicator reaction is abolished or attenuated by SOD. The generation of °2'- via enzymatic, chemical, electrochemical, or photochemical methods as well as by ionizing radiation (see 20, 21. 128). is usually coupled to spectrophotometric obser­ vations of changes in the detector molecule accompanying the above redox reactions (120, 128). Other methods of detection include NADPH oxida­ tion in the presence oflactate dehydrogenase (18), spin-trapping coupled to electron spin resonance (ESR) measurements (65), or direct observation of O2'- decay rates (21, 196). All the above systems and methods have certain advantages and disadvantages-the most suitable detector system has to be selected for each individual purpose and biological system under investiga­ tion. Hydrogen Peroxide (H202) Similar to O2'-, H202 can act both as an oxidant and a reductant; however, in the absence of metal catalysts or enzymes it has a low reactivity toward most organic molecules. Its formation can be detected by direct spectropho­ tometric methods, coupled (color) reactions in the presence or absence of peroxidases [for example NADH-peroxidase (43)], polarographically as an increase in oxygen tension upon addition of catalase, or radiochemically by the decarboxylation of p4C a-keto acids (205). The latter two methods are especially suitable for turbid solutions (44, 47, 79). A nn u. R ev . P la nt . P hy sio l. 19 82 .3 3: 73 -9 6. D ow nl oa de d fro m w w w .an nu al re vi ew s.o rg by Z he jia ng U niv ers ity on 08 /11 /12 . F or pe rso na l u se on ly. 78 ELSTNER The OH Radical OH' is believed to be the active species in reactions inhibited by both SOD and catalase, and is formed via the catalyzed Haber-Weiss cycle: O2'- + MO+-02 + M(o-l)+ lOa. HzOz + M(o-l)+ -OH-+OH + MO+ (17, 33, 85, 87) lOb. Overall reaction: Oz'- + HzOz-OH- + OH' + Oz. to. As can be seen from reactions lOa and lOb, the formation of OH' is dependent on both HZ02 and 02'- and thus subject to inhibition by both SOD and catalase. Metal (M) catalysis is necessary since the rate of the uncatalyzed overall reaction 10 is negligible (19, 20, 164). It has been proposed that the product of reactions 10 a,b might be responsible for generally random destruction (20). The short lifetime and the strongly positive redox potential (close to + 2 V) of "free" OH' render its sites of reaction close to its point of f ormation (19, 20, 197). In this context, organic oxygen radicals (alkoxy, peroxy), semiquinones, reduced hydrogen perox­ ide, hydrogen peroxide-electron donor complexes (crypto-OH), as well as metallo-oxygen complexes have been proposed as the ultimately active species instead of the randomly destructive free OH ' (19, 20, 57, 59, 89,131, 156,198,204). We probably have to envisage the existence of both free OR" formed via an iron-catalyzed Haber-Weiss cycle (10 a,b) as an unspecific, extremely aggressive species, and reduced HzOz (electron donor-HzOz com­ plex) together with organic radicals as more specific, but still very reactive, oxygen species. As already mentioned, the detection of free OH radicals (or similarly reactive equivalents) is based mainly on the inhibition of a detector reaction by both SOD and catalase, as well as by (unspecific) OR" Scaven­ gers such as benzoate, formate, mannitol, ethanol, a.-tocopherol and others, which also react at approximately equal rates with '02, Examples of detec­ tor reactions include ethylene formation from methional (17), spin trapping (65, 90), and bleaching of p-nitroso-dimethyl aniline (19) in context with certain inhibitors (33). Singlet Oxygen '02 is formed by photodynamic processes through interactions of several oxygen radicals (for example, during lipid peroxidation) and by electron donation from Oz'- to certain electron acceptors (68, 69, 78, 117, 119, 176, 180, 182, 183). The detection of '02 has been based on (unspecific, see above) detector reactions for 'Oz with the aid of nonspecific 'Oz quenchers or scavengers, or by bioluminescence. None of the applied methods on its own is a reliable indicator for the detection of 102, with the possible excep­ tion of 7-0H cholesterol formation (in contrast to 5-0H cholesterol forma­ tion by radical attack) from cholesterol in the presence of 'Oz (68, 69, 117). A nn u. R ev . P la nt . P hy sio l. 19 82 .3 3: 73 -9 6. D ow nl oa de d fro m w w w .an nu al re vi ew s.o rg by Z he jia ng U niv ers ity on 08 /11 /12 . F or pe rso na l u se on ly. ACTIVATED OXYGEN 79 Organic Peroxides and Peroxy Radicals As stated above, it is difficult to differentiate bet)Veen reactions involving OH radicals and organic alkoxy and/or peroxy radicals, since these species are interrelated in biological systems by reactions with rate constants close to diffusion limitations (20, 178). Cooxidation of carotenoids (82, 181), determination of breakdown products [methylene oxindole from indolea­ cetic acid (200), malondialdehyde, and/or ethane fr9IY �nsaturated fatty acid (12, 36, 109, 116)] only indicate that these reactions in toto include intermediary peroxy and/or alkoxy radicals (131, 191). OXYGEN ACTIVATION IN DIFFERENT COMPARTMENTS AND ORGANELLES OF PLANT CELLS Peroxides in Cell Walls The lignification of cell walls involves the syntpesis of the phenylpropanoid precursors of lignin, and H202 is required for their subsequent polymeriza­ tion (83). The importance of peroxidase(s) together with H202 for lignifica­ tion has been shown cytochemically (91, 103) as well as via direct demonstration of H202 production by isolated cell walls (47, 84, 88). The oxygen activating reactions observed in cell walls have previously been described (199) and have been shown to involve monophenols, Mn2+, peroxidase(s), and NADPH as the electron donor. Reduced NADP is probably derived from the activity of a cell wall-bound malate dehydroge­ nase (84). A reaction scheme involving the above mentioned steps (together with O2'- as an intermediate) as well as the resolution of three cell wall­ associated and/or bound peroxidases has been presented (84, 126). An interesting area of speculation regards the involvement of the above men­ tioned oxygen activation in cell walls in the defense against parasites, since there are similarities with oxidative killing in phagocytic cells (14a, 165). Microsomal Oxygen Activation The biochemical criterion of microsomes (i.e. the microsomal fraction after differential centrifugation of cell homogenates) is an electron transport system composed of fiavoproteins, nonheme iron proteins, and cyto­ chromes, e.g. cytochrome P4S0' NADPH serve(s) as electron donor(s) (194). The reduced metallo-oxygen complex (P 450) is the active species in the mixed function system according to RH + NADPH + H+ + O2 ---� ROH + NADP+ + H20 11. Microsomal preparations have been obtained from animal tissues, plants, and microorganisms exhibiting different oxidative activities such as hy­ droxylations (cf reaction 11), fatty acid desaturation, initiation of lipid A nn u. R ev . P la nt . P hy sio l. 19 82 .3 3: 73 -9 6. D ow nl oa de d fro m w w w .an nu al re vi ew s.o rg by Z he jia ng U niv ers ity on 08 /11 /12 . F or pe rso na l u se on ly. 80 ELSTNER peroxidation, and demethylations (96, 106, 180). In higher plants, p-hydroxylation of