Biochemical Effects of Excited State Molecular Oxygen
Jeffrey Bland University of Puget Sound Tacoma. Washington 98416
The extent to which the excited state molecular oxygen species, singlet oxygen [02('Ag)], participates in hiochemical processes is becoming better understood. Recent research has implicated singlet 0 2 reactivity in many cellular events. Biochemical roles of excited state oxygen include its presence: 1) in dye sensitized photooxidations (photodynamic action), 2) in certain blood diseases, 3) in cancer inducing mechanisms, 4) in possible free radical-like aging mechanisms, 5) in the role of the bactericidal activities of phagocytes, and 6 ) in metabolic hydroxylations. This report is designed to introduce current research which illustrates the intimacy of singlet 0 2 in these six areas, and demonstrate the general role of singlet oxidation in biochemical processes. Physical and Chemical Nature of Singlet Oxygen T o fully appreciate the reactivity of singlet state oxygen it is necessary to understand the electronic structure of the oxygen molecule. The two highest energy electrons in ground state 0 9 are in deeenerate oi molecular orbitals. whereas in singlet 0 2 the highest energy electrons are in the same orbital (a A state because their anmlar momentum is in the same direction) and their spin opposed (denoted by a superscript 1). A second excited state of oxygen is encountered when the highest energy electrons are in different orbitals (a L: state because their angular momentum is opposite), and their spins opposed ('Z). The two excited states of oxygen can thus be represented as in the following diagram (I). Orbital Occupancy
Energy above
Stare
Symbol
groundstate
2nd Excited 1st Excited Ground state
1Z
37 kcai 22 Kcal
1A 3Z
1# 1-
4-
1-
The lifetime of the ' 2 state is very short, lo@ s, and is thought to decay to the 'A state before appreciable chemistry can occur (2). The lifetime of the 'A state in condensed phases is highly solvent dependent, and on the order of 10-"10-5 s in aqueous systems (3). Relaxation of singlet oxygen to the ground state can occur with the production of the so-called "dimol" chemiluminescence a t 6334 A and 7032 A (4). Singlet oxygen can be produced via a variety of chemical and physical means as illustrated in the table. The reactivity of singlet 0 2 resembles that of ethylene; however, singlet 0 2 is a more electrophilic reactant. I t reacts very quickly with hydrocarbons containing unsaturation via three general mechanisms. Exposure of 1,3dienes to singlet oxygen leads to the thermally allowed 4 Methods of Production of Singlet 0 ,
2 dioxetane cycloaddition product (12, 13).Many of these dioxetane products have been isolated, and in some cases have been found to be moderately stable crystalline solids (14, 15). This mode of addition of singlet oxygen to dienes has been suggested to be important in the air oxidation of resin acids and munoterpenes such as levopimaric acid (16), ar-phellandrene (17), and a-terpinene to yield ascaridole (18),a naturally occurring dioxetane.
Exposure of compounds containing isolated double bonds leads to the production of allylic hydroperoxides via an "ene" type reaction (19). Lastly, some " 0 2 " hydrocarbon reactions may be considered hydrogen abstraction processes (20).
Exposure of compounds containing heteroatoms such as nitrogen, or sulfur, often leads to oxidation of the heteroatom (21,221.
+
Chemical Methods sodium hyPachioridehydrogen peroxide (51 tripnenyl phorphene oronide (61 1-pholpna-2.8.9-tr10xaadamantane azonide (71 superoxide anion (81
~+ NaOCC1O, 0 % + NaCl + H,O
~
8 m3
0
\"/
+&Po
+
"
2 H + + 20,--'02
+ H*O>
Physical Methods ohotosenritizatian(1, 9 ) (Photodynamic conaitionr) microwave discharge (101 atmuspheric generation (771
0 + senr*+'o,
oz+ h w 1 0 2
274 / Journal of Chemical Education
+ Senr
The classes of different hiomolecules which have been found to react readily with oxygen under dye sensitized photooxidation conditions include amino acids (23), proteins and enzymes (24-26), nucleic acids (27-291, and membrane components (30;31). There has been much concern that the reactive species under these conditions is not a free singlet 02, hut rather involves the production of free radicals or radical ions by interaction of the sensitizer trip-
let with a reducing suhstrate in the following manner (the so-called Type I process)
Two methods have been employed to determine the extent of involvement of free singlet 0 2 in these processes. The first is to generate singlet oxygen by an unambiguous method such as microwave discharge and compare the products of this oxidation with those of the corresponding dye sensitized photooxidation. Fischer (32) has compared the products from photooxidation and microwave discharge mediated oxidation of amino acids and found them to he the same, suggesting free ' 0 2 in the dye sensitized oxidation. Oxidation of lysozyme (33) by both techniques also gave identical oxidation products. Work by Knowles (34, 3.5), indicates, however, that when the substrate has a high oxidation potential it many times gives different products depending upon the oxidation method. Oxidation of guanosine (36, 37), yields two different products depending upon whether the oxidation is carried out in the presence of methylene blue or via microwave discharge. The second method used to test the presence of free singlet 0 2 is by using systems which shorten or lengthen the effective lifetime of singlet 0 2 . Merkel, Nilsson, and Kearns (38) have shown that the lifetime of singlet 0 2 is 10-fold longer in D 2 0 than it is in HzO. This enhanced singlet 0 2 lifetime in D20 can he used as a test for singlet 0 2 presence in dye sensitized oxygenations. The same strategy has been employed by Nilsson and Kearns (39) using azide ion which has been found to be a selective ' 0 2 product quencher. Using these methods, reports suggesting the intermediacy of free singlet 0 2 in dye sensitized photooxidation of proteins and amino acids have appeared ( 4 0 4 2 ) . Recently it has become apparent that there is another potentially important source of singlet Oz in hiochemical systems, that which may he produced via dismutation of superoxide radical anion 2H+
+ 20,-
-
'0, or
'0,+ HAI
This dismutation process can occur chemically (43) or be catalyzed by the enzyme superoxide dismutatase (erythrocuprein) (44). Superoxide anion has been found to he formed bv manv. orocesses in biolonical . - systems such as in dye photosensitized oxidations, reduction of oxygen hy enzvmatic cofactors such as flavin derivatives, nicotine amide derivatives or quinones, and enzymatic oxidoreductases (45). The enzyme superoxide dismutase (SOD), a metallprotein containing 2g-atoms of Cu and Zn each, catalyzes the dismutation of 02-. to give molecular oxygen. The question as to whether the oxygen produced is in the ground or singlet excited state after enzymatic dismutation has recently been resolved by Meyeda and Bard (46). Singlet oxygen produced by chemical dismutation of superoxide was effectively trapped as the 4 2 cycloaddition product using 1,3-diphenylisobenzofuran.
+
A? AF = C.H.
In the presence of SOD, however, the amount of addition product was greatly reduced demonstrating that SOD interferes with singlet 0 2 production by dismutating superoxide to ground state oxygen. The only other explanation of these results is that SOD is acting as a singlet 0 2 quencher. Schaap et aL7, using singlet 0 2 generated by
photosensitization, demonstrated that SOD is not an effective singlet 0 2 quencher. They suggest that SOD protects hioloeical svstems from oxidative damape via singlet oxidation by dismutatlng superoxidc to grotl;~d srate ¥ hefore ir rhemicall\. aismutntes to singlet 0,. It is inreresting to note that SOD is not denatured under photooxygenation conditions which are known to denature many enzymes. Superoxide anion radical is known to undergo chemical dismutation in the Dresence of a radical cation R.+ (ex., ferricenium cation) tb yield singlet oxygen (47). It is not unreasonable to conclude that superoxide anion once produced in a biochemical system is partitioned between two fates: one being enzymatic dismutation to ground state oxygen and hydrogen peroxide, and the other chemical dismutation to singlet 0 2 . If SOD is working in an environment which experiences singlet 0 2 produced by chemical dismutation, then it is consistent with its proposed role as a protective agent against superoxidation that it he somewhat resistant to singlet oxidation. Singlet Oxygen lnvolvemenl in Dye Sensitized Photooxygenations Since Raah first exposed paramecia to acridine and noted that they were killed only in the presence of light, photobiological phenomena in terms of photosensitization by dyes and pigments has been an active area of research (48). Several reviews have assessed the importance of dye sensitized photooxidation or photodynamic action in hiological processes (9, 49, 50). It is now recognized that porphyrin containing molecules such as chlorophyll and hemoglobin are capable of sensitizing oxygen to its excited singlet state. Foote (51) has shown that this oxidant species behaves chemically similar to singlet 0 2 produced by the hydrogen peroxide-hypochlorite system, thereby implicating singlet 0 2 as the oxidant in photodynamic processes. Foote, Chang, and Denny (52) have suggested that plants are protected from oxidative damage by oxygen sensitization from their own chlorophyll due to the presence of carotenoid pigments which are excellent singlet oxygen quenchers. Mutants lacking carotenoids are killed in the presence of light and oxygen. Other typical biological quenchers of singlet 0 2 include riboflavin, vitamin A, and phaeophytin-a. Alpha-tocopherol has been found (53) to ieact chemically with singlet oxygen, thereby presenting itself as a potential chemical trap. Morphological evidence for photodynamic damage to organisms has heen observed to include membrane damage, mutagenesis, and changes in renroduction. There are a number of ~hotodvnamicdiseases'which are found in animals and man, including dietary photosensitizer effects upon grazing animals (48) or certain types of drug photosensitivity in man (54). Singlet Oxygen Involvement in Blood Disease Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is associated with a host of defects in erythrocyte metabblism, and stands as the most common genetic abnormality in man (55). In all cases the erythrocytes are extremely sensitive to oxidative processes. Recent research has emphasized the importance of oxidation protecting enzyme systems such as superoxide dismutase (56) and catalase (57) in protecting against blood hemolysis. Luhin, Fromm, and Oski (58) report evidence for involvement of hemoglobin-bound oxygen in the hemolysis of vitamin-E deficient erythrocytes. Collman has recently demonstrated that in their model of the oxygen bonding site in oxygen transport molecules the oxygen was complexed perpendicular to the porphyrin ring, and had a temperature-dependent Oz stretching frequency which resembles a coordinated singlet oxygen more closely than a coordinated groundstate oxygen (59). These results suggest the possible involvement of singlet oxygen in the erythrocyte dysfunctions associated with GGPD deficiency either via chemical Volume 53, Number 5, May 1976 / 275
dismutation of superoxide or with a hemoglohin-coordinated singlet oxygen. These processes are undoubtedly also dependent upon other oxidant species such as hydrogen pe;oxide, hydroxyl radical, and superoxide itself; yet the potential importance of singlet 0 2 merits further study. A group df hlood diseases termed porphyria are associated with acute photosensitivity (60). These include porphyria cutanea Grda, variegate porphyria, and erythropoietic protoporphyria (EPP). E P P is an inherited disorder whereby the erythrocyte is characterized as having a high concentration of free protoporphyrin. Harber, Fleischer, and Baer (61) demonstrated that long uv irradiation of red cells obtained from E P P patients produced hemolysis. More recently Goldstein and Harber (62) have demonstrated that red cell memhrane damage associated with ohotohemolvsis occurs concomitant with lipid peroxida. . tioo. The intermediacy of singlet oxygen in this membrane destructive process characteristic of E P P was shown by Lamola, ~ a m k e and , Trozzolo (63). They used &carotene, the effective singlet oxygen quencher, and were able to prevent lipid hydroperoxide formation in irradiated blood from E P P donors. Bland, et al. (64) have recently shown that a-tocopherol, a singlet O2 sink, also effectively reduces erythrocyte photosensitivity in uiuo. Photoerythema has also been successfully treated clinically by oral doses of 8carotene (65). These results all point to the fact that singlet 0 2 is important in photoinduced erythrocyte hemolysis, and that this hemolysis is induced by cell membrane photoperoxidation via singlet oxygen which weakens the membrane and results in lysis of the erythrocyte. Role of Singlet Oxygen in Cancer Inducing Processes The literature contains numerous examples associating the photosensitizing ability and carcinogenicity of polynuclear aromatic hydrocarbons (48, 66, 67). Two theories implicating singlet 0 2 with hydrocarbon carcinogenicity have been proposed. Kahn and Kasha (68) have proposed the "optical residue" theory in which the polynuclear aromatic is bound to a cellular constituent, and undergoes excitation which then generates singlet 0 2 . The excited oxygen thus produced is then capable of intracellular damage leading to tumor initiation. The alternate theory of Steele, et al. (69) differs from that of Kasha and Kahn. The singlet oxygen once producpd reacts w i ~ hthe K region of rhe hgdrocarhon prudllrina an aromatic hydroperuxide. These hydroperoxides might act as masked singlet 0 2 sources because their decomposition is known to produce excited 0 2 . Hydroperoxide formation, however, is not a facile reaction of these hydrocarhons, and it would appear as if this latter process is not as likelv. Foote (70j h a s demonstrated that excited hydrocarhons can he nroduced in the absence of liaht: therefore, it might he pos&hle for these to sensitize singlet 0 2 productionin the dark. Harman (71) has found that the singlet O z trap a-tocopherol exhibits an in uiuo inhibiting effect on dimethylhenzanthracene induced carcinogenesis, supporting the potential role of singlet oxygen or its products in these processes. It has been reported (72) that malignant cells take up and bind the singlet O2 sensitizer hematoporphyrin to a areater extent than normal tissue, and that irradiation selectiwly kills rhe tumor cells due possibly tn preferential sinclet mypen exposure. hlore recentlv a report proposing a mechanism which links skin cancer with photo sensitization has appeared (73), further implicating singlet 0 2 in carcinogenicity. Chan and Black suggest that the photochemical conversion of cholesterol to its carcinogenic 5a, 6a epoxide is a possible mechanism for sunlight induced epidermal carcinogenesis. The enzyme cholesterol 5,6-epoxidehydratase which deactivates the carcinogenic sterol can he induced by exposure of the organism to light. The photoproduced oxidant species could he either superoxide, singlet 0 2 , or their secondary oxidant products. 276 / Journal of Chemical Education
These theories associating singlet 0 2 with carcinogenesis remain hiahlv . yet . merit attention as potential - -speculative, . sources of carcinogenic biochemicals in uiuo. Role of Singlet Oxygen in Free Radical-Like Aging Processes Aging has long been associated with free radical production during the course of normal cellular metabolism (74, 75). Aging of the skin is correlated with oxidative polymerization of elastin which might be a singlet O z mediated process. Most recently singlet oxidation has been found to be involved in lipid peroxidation when generated by the xanthine oxidase (75) or lipoxidase (77) enzyme systems. Singlet O1 is known to react with sites of unsaturation to proiuce iydroperoxides. These hydroperoxides are excellent free radical oroaenitors. and lead to radicals upon hydro. gen atom abstraction by a suitable biochemical hydiogen acceptor aeent such as superoxide radical anion. The mechanism hy which free radicals produce their effects on cells is not well understood. I t is possible that cellular dysfunction may result from adventitious compartmentalization of cellular materials throuah cross-linking of nearby macromolecules by hydroperoiy radicals (78). Changes in membrane characteristics of mitochondria and lysosimes due to lipid peroxidation have been found hy several workers (63, 64, 79, 80). Lipofuscio, fluorescent lipid containing granules, is known to accumulate in the heart, brain, and muscle of aged persons (81). The insoluhility of lipofuscin leads to long-term accumulation of iudigestible damaged memhrane material. Recently lipid peroxidation and related free radical intermediates have been demonstrated to be critical in the reactions initiating the formation of lipofuscin. Zimmerman, et al. (82) have found that lipid peroxidation is inhibited by superoxide dismutase in the isolated inner memhrane of rat liver mitochondria. This could in part he due to the prevention of chemical dismutation of superoxide anion to singlet oxygen, thereby preventing lipid peroxidation. The accumulation of linofuscin has been imolicated to be involved in the deficiency of memory and learning functions. Puri, Pogarcar, and Lal (83) . . showed that rats on a chronic vitamin E deficient diet accumulated lipofuscin and exhibited poor memory and learning skills. Packer and Smith (84) working with human fibroblasts have found that vitamin E increased life span to twice the number of douhliogs. These results are consistent with a model whereby singlet 0 2 if not trapped by a biological antioxidant is capable of leading to lipid peroxidation and the formation of lipofuscin bodies which results in aging of the system.
-
Role of Singlet Oxygen in Phagocylosis In 1967 Klehanoff (85) showed that phagocytosis, the process by which some types of white hlood cells such as polymorphonuclear leukocytes engulf and destroy microorganisms. is mediated hv hydrogen perox. mvelo~eroxidase, - . yde, and'halide ions. Subsequent work has &own that neither myeloperoxidase nor halide ions exhibit microhicidal activity, and the activity of hydrogen peroxide is much less than that of the complete system. Recent work has implicated superoxide and-singlet oxygen as additional microhicidal agents in phagocytosis. Holmes, et al. (86) found that patients with chronic granulomatous disease, a syndrome characterized by defective hactericidal activity, do not produce quantities of superoxide anion. The neutrophils from patients afflicted with this syndrome were shown to phagocytize bacteria normally, yet were unable to kill them. The defect therefore was postulated to he in the release of the bactericidal superoxide. I t was subsequently found (87) that the enzyme superoxide dismutase was missing from the cytosol of neutrophils of normal humans. Fridovich (88) suggests that the absence of this enzyme might enhance the hactericidal capacity of these cells supporting
the role of superoxide in phagocytosis. Johnson and DeChatelet (89) have recently challenged this position in that they find considerable SOD activity in the cytosol of neutrophils. Recent reports (90, 91), however, continue to implicate superoxide anion in the action of leukocytes. The involvement of excited state oxygen in phagocytosis was first proposed by Allen, Stjernholm, and Steele (92) in which they reported chemiluminescence from oxetane cleavaee in the dieestion of eneulfed microoreanisms hv polymurphon~lrlearleukwytes. They found the intensity id the emitred lieht to be . DrnDurli0nal lo the IellkoclTe'l ely. " . colytic activity in the hexose monophosphate shunt therebv huildine NADPH reducine Dower which is the most important source of energy req;'ired for phagocytosis. They also found no chemiluminescence from leukocytes of children suffering from chronic granulomatous disease. Fridovich (88) argues that chemiluminescence is not proof of singlet 0 2 invdvement in that many chemical reactions, particularly those involving free radicals, can produce chemiluminescence. Recently Porter and Iugraham (93) have found that the decomposition of hydrogen peroxide by catalase, an enzyme mediated reaction thought (94) to generate singlet 0 2 due to the observed chemiluminescence, was found to oroduce
