Proc. Nat. Acad. Sci. USA Vol. 68, No. 5, pp. 1024-1027, May 1971 An Enzyme-Based Theory of Obligate Anaerobiosis: The Physiological Function of Superoxide Dismutase JOE M. McCORD, BERNARD B. KEELE, JR.*, AND IRWIN FRIDOVICH Department of Biochemistry, Duke University Medical Center, Durham, North Carolina,27706 Communicated by Philip Handler, February 11, 1971 ABSTRACT The distribution of catalase and super- not contain catalase, e.g., the streptococci, pneumococci, and oxide dismutase has been examined in various micro- lactic acid bacteria. More recently, Agromyces ramnosus, the organisms. Strict anaerobes exhibited no superoxide dis- mutase and, generally, no catalase activity. All aerobic predominant soil organism, was found to lack catalase, de- organisms containing cytochrome systems were found to spite its aerobic metabolism (8, 9). Further, some strict contain both superoxide dismutase and catalase. Aero- anaerobes have catalase activity, yet cannot tolerate ex- tolerant anaerobes, which survive exposure to air and posure to air (10). metabolize oxygen to a limited extent but do not contain cytochrome systems, were found to be devoid of catalase The recent discovery and characterization of superoxide activity but did exhibit superoxide dismutase activity. dismutase (11, 12) raised the question of its physiological This distribution is consistent with the proposal that the role. The substrate, a reactive and potentially detrimental prime physiological function of superoxide dismutase is free-radical form of oxygen, had long been implicated as an protection of oxygen-metabolizing organisms against the intermediate in the reduction of 02 by a family of metallo- potentially detrimental effects of the superoxide free radical, a biologically produced intermediate resulting flavoenzymes (13-17). Evidence that the superoxide radical is from the univalent reduction of molecular oxygen. released into free solution from the enzyme surface was not obtained until 1968 (11), but was then quickly confirmed for Pasteur's discovery that certain organisms are not only capable the xanthine oxidase system by the detection of its electron of growing in oxygen-free environments, but in many cases paramagnetic resonance signal (18). The reaction catalyzed are restricted to such environments, has never been satis- by superoxide dismutase, factorily explained. Obligately anaerobic organisms are strongly inhibited or killed by exposure to molecular oxygen 02- + 02- + 2H+ - 02 + H202, (1). The possible role of catalase in protecting aerobic micro- organisms from death by hydrogen peroxide poisoning was proceeds spontaneously at pH 7.7, with a rate constant of ap- rather quickly recognized. In 1893 Gottstein discovered that proximately 2 X 105 M-I sec- (19). The ubiquity and con- certain bacteria decomposed H202 with the liberation of a gas stancy of this enzymic activity in a wide variety of tissues and (2). In 1907 it was observed that certain anaerobic bacteria organisms led us to seek its physiological importance. Interest contain no detectable catalase, whereas all the aerobes ex- was heightened by the discovery that the superoxide dismutase amined exhibited significant catalatic activity (3). This led of Escherichia coli is a manganoprotein (20), which bears little to proposals that oxygen toxicity was occasioned by its re- resemblance to the copper- and zinc-containing mammalian duction product, hydrogen peroxide (4, 5). Proceeding from enzyme (12, 21), but which is nevertheless present in the or- this assumption investigators reasoned (4) that: (a) H202 ganism at similar concentration and displays a nearly identical should accumulate in aerobic cultures of anaerobes, as a result specific activity. either of bacterial metabolism or of the action of light on the The data presented in this report strongly implicate medium, (b) anaerobes should be sensitive to externally added superoxide dismutase as being vital to the existence of any hydrogen peroxide and, (c) anaerobes should grow aerobically organism that metabolizes oxygen. when catalase is present in the medium. Technical limitations at the time prevented detection of the low but toxic concentra- MATERIALS AND METHODS tions of H202 that anaerobes produce. It was, however, subse- Frozen or lyophilized cells of certain microorganisms were quently shown that nearly all anaerobes do produce H202 (6). generously supplied for this study as follows: Salmonella The sensitivity of anaerobes to H202 was readily shown, and a typhimurium and an unidentified pseudomonad from Dr. high degree of variation was apparent (4, 7). It could not be Henry Kamin; Halobacterium salinarium from Dr. Jayant shown that the presence of catalase would allow aerobic Joshi; Rhizobium japonicum from Dr. Gerald Elkan; My- growth of anaerobes (4, 5), although one anaerobe grew cobacterium sp. from Dr. Jerome Perry; Veillonella alcalescens better at low oxygen tension with catalase present (4). Thus, and Butyribacterium rettgeri from Dr. Charles Wittenberger; although catalase aids the survival of some microorganisms Clostridium pasteurianum, Clostridium sticklandii, Clostridium in aerobic media, catalase activity does not provide a suf- lentoputrescens, Clostridium barkeri, and Clostridium sp. ficient answer. Many organisms capable of aerobic growth do (strain M.E.) from Dr. T. C. Stadtman; Butyrivibrio fibri- solvens from Dr. Sam Tove; Clostridium cellobioparum and * Present address: Institute of Dental Research, University of N2C3, an unclassified rumen organism, from Drs. Robert Alabama Medical Center, Birmingham, Ala. 35233. Mah and R. E. Hungate; Zymobacterium oroticum from Dr. 1024 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Theory of Obligate Anaerobiosis 1025 K. V. Rajagopalan; and Clostridium acetobutylicum from TABLE 1. Superoxide dismutase and catalase contents of a Mr. Robert Waterson. E. coli cells were obtained as a frozen variety of microorganisms paste from Miles Laboratories. Micrococcus radiodurans, Saccharomyces cerevisiae (ATCC 560), Streptococcus fecalis, Superoxide Streptococcus mutans, Streptococcus bovis, Streptococcus mitis, dismutase Catalase Streptococcus lactis, and Lactobacillus plantarum were grown (units/mg) (units/mg) by us on either trypticase soy broth or APT broth, available Aerobes: from BBL, Cockeysville, Md., or on Brain-Heart Infusion Escherichia coli 1.8 6.1 Broth obtained from Difco. Cultures were obtained as follows: Salmonella typhimurium 1.4 2.4 M. radiodurans from Dr. Jane Setlow; S. fecalis and L. plan- Halobacterium salinarium 2.1 3.4 tarum from Dr. John McNeill; and S. mutans 6715, S. lactis Rhizobium japonicum 2.6 0.7 (ATCC 19435), S. bovis (ATCC 9809), and S. mitis (ATCC Micrococcus radiodurans 7.0 289 9811) from Dr. James Sandham. Saccharomyces cerevisiae 3.7 13.5 Cells were suspended in 50 mM potassium phosphate Mycobacterium sp. 2.9 2.7 buffer, pH 7.8, containing 0.1 mnM EDTA, and sonicated Pseudomonas sp. 2.0 22.5 in an ice bath by means of a Branson sonifier at a power Strict Anaerobes: setting of 100 W to disrupt the cells. After centrifugation, Veillonella alcalescens 0 0 the cell-free extracts were assayed for superoxide dismutase Clostridium pasteurianum, sticklandii, activity as previously described (12). In certain cases, the lentoputrescens, cellobioparum, presence of low molecular weight substances capable of barkeri 0 0 reducing cytochrome c necessitated an overnight dialysis Clostridium acetobutylicum 0 of the cell-free extracts to remove these interfering substances. Clostridium sp. (strain M.C.) 0 0 The extracts were assayed for catalase activity by means of Butyrivibrio fibrisolvens 0 0.1 a Gilson Oxygraph equipped with a Clark electrode and a N2C3* 0 <0.1 thermostatted cell. The assay mixture contained 0.02 M Aerotolerant Anaerobes: hydrogen peroxide in 50 mrM potassium phosphate buffer at pH 7.8, containing 0.1 mM EDTA, at 25°C. Rates of Butyribacterium rettgeri 1.6 0 Streptococcus fecalis 0.8 0 oxygen production were compared to the rate obtained with Streptococcus mutans 0.5 0 a standardized solution of bovine liver catalase obtained Streptococcus bovis 0.3 0 from Sigma. Alternatively, catalase was assayed spectro- Streptococcus mitis 0.2 0 photometrically by the method of Beers and Sizer (22). Streptococcus lactis 1.4 0 Protein contents of cell-free extracts were estimated at 280 Zymobacterium oroticum 0.6 0 nm assuming Elm (c = 1%) = 10.0. Lactobacillus plantarum 0 0 RESULTS AND DISCUSSION * N2C3 is an unclassified cellulolytic Gram-negative rod is5- The superoxide dismutase and catalase contents of 26 species lated from the rumen of an African zebu steer and has been of microorganisms of three categories are reported in Table described by Margherita, S. S., and R. E. Hungate, J. Bacteriol., 1. The aerobes are the microorganisms that can utilize molec- 86, 855 (1963). ular oxygen as the terminal electron acceptor. The obligate anaerobes not only lack the cytochrome system necessary The strict anaerobes for aerobic respiration, but are unable to survive under conditions of aeration. The organisms termed aerotolerant The ten organisms in this classification represent various anaerobes do not utilize molecular oxygen as the terminal clostridial species and three organisms that were isolated electron acceptor for their energy metabolism, but, with originally from the bovine rumen. Aeration is lethal for all one exception, they are capable of reducing oxygen to a these species. Strict anaerobes are nearly always