Photochemistry and Photohiology, 1977, Vol. 26, pp. 4147.
Pergamon Press. Printed in Great Britain
THE SUPEROXIDE ANION AS ELECTRON DONOR TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN H. NINNEMANN*, R. J. STRASSER~ and W. L. BUTLERS Department of Biology, University of California at San Diego, La Jolla, CA. 92093, U.S.A. (Received 24 December 1976; accepted 4 February 1977)
Abstract-In isolated respiratory multienzyme complexes of beef heart mitochondria the b-type cytochromes can be photoreduced in presence of flavin via the superoxide anion. 0; does not reduce cytochrome c,. In an anaerobic system, FMNH, formed by irradiation with blue light in presence of EDTA reduces cytochromes b and c l . The possible implication of 0; in the electron transfer from flavin/flavoprotein to cytochrome b in' blue light-controlled biological processes is discussed. INTRODUCTION
well, either by the substances of interest, such as proteins serving as the electron donor for the photoreduction of flavin, or via the hydrogen peroxide generated by the dismutation of 0;.
Superoxide anion is commonly formed in cells or organelles that utilize oxygen. Generally 0; is regarded as being toxic for biological systems either by acting directly as a strong oxidant or reductant, MATERIAL AND METHODS or possibly indirectly by forming H,O, or the exMultienzyme complexes 1-111 (NADH-cytochrome c tremely reactive hydroxyl radical OH'. NADPH- reductase) and 111 (hydroquinone-cytochrome c reductase) dependent peroxidation of microsomal lipids (Peter- were prepared from beef heart mitochondria following the son and Aust, 1972), glutathione-induced swelling of procedure of Hatefi et al. (1961, 1962). The complexes were mitochondria (Levander et al., 1974), glutathione- suspended in TSH buffer at pH 8.0 (0.05 M Tris-CI, 0.66 M sucrose, 1 mM histidine). The superoxide anion 0; was induced lipid peroxidation in isolated inner mito- generated photochemically by irradiating FMN (10 or chondrial membrane fragments (Zimmermann et al., lOOpM) in the presence of EDTA which had been added 1973) are a few of those reactions which seem to in- to the multienzyme complexes. Irradiation was performed with a prefocused Xe lamp volve 0 2 and can be prevented by superoxide dismuEimac Division, VIX-150) in combination with a tase (SOD). There are also reports on 0; serving (Varian, 460 nm interference filter (Balzer) and 2.5 ern CuSO, soluas electron donor to a solution of ferri-cytochrome tion (15%) resulting in 4 W/mz. Within seconds after the c (McCord and Fridovich, 1968; 1969; Land and irradiation, absorption spectra were measured at room Swallow, 1971). This reaction has been used as a temperature with a Cary 14 spectrophotometer (tungsten simple assay for the presence of the superoxide anion. lamp, EM1 9558C phototube and logarithmic photometer) on line with a Digital PDP/8 computer with a 12-bit We wish to report experiments which show that analog-to-digital converter. The single beam absorption irradiation of mitochondria1 multienzyme complexes spectra were stored in the memory core of the computer 1-111 and 111 of beef heart mitochondria supple- and displayed as difference spectra between sample and mented with soluble flavins and an electron donor reference. Simultaneous measurements of absorption changes of to the flavin resulted in a steady uptake of oxygen cytochrome b at 560 nm (AA 560), of changes in flavin fluat a rate which depended on the intensity of light orescence (measured at 609 nm, AF 609) and of the oxygen and the concentration of flavin. The 0; formed in content of the enzyme system were performed with an optithese reactions served as a selective electron donor cal set-up constructed in this laboratory (R. Strasser) to the b-type cytochromes of the respiratory electron (Fig. 1). The measuring beam (MB) from a tungsten iodine lamp (lamp 1) passed through two Corning cut-off transport chain. Irradiating the flavin-supplemented filters 3486, an interference filter S1 with T,,,= 560nm multienzyme complexes under anaerobic conditions and through a Bausch and Lomb monochromator (MC 1) led to the photoreduction of the flavin and, under set at 560 nm for measuring cytochrome b absorption these conditions, the reduced flavin reacted directly changes. It then passed a light pipe (Schott, Mainz, W. Germany), a light mixing rod (LMR) and an inverted lightto reduce the cytochromes b in suspension. In addi- transmittant oxygen electrode (Clark type, LOE; Strasser, tion to these flavin-mediated photoreductive reac- 1974). The sample S (loo@, 1 mm thickness) was placed tions, photooFidative reactions may be generated as between this 0,-electrode and a second light mixing rod *On leave of absence from the University of Tubingen, W. Germany. Supported by the Deutsche Forschungsgemeinschaft by grants No. Ni 119/4 and 5. Present address: Institut fur Chemische Pflanzenphysiologie, CorrensstraBe 41, 7400 Tubingen, W. Germany. ?Supported by the Swiss National Fonds. $Supported by US. Public Health Service grant GM 20648. 41
(LMR). Here the measuring light was picked up behind the sample, guided through a filter combination of Corning cut off filter 3486 and Balzer interference filters 557 and 562nm to a Hamamatsu phototube R818 (PM1). The absorbance changes were plotted on a Hewlett Packard 7074A recorder where off-setting and proper amplification allowed a full scale sensitivity of 0.008 A per 20 cm. The actinic light from a high intensity mercury lamp (lamp 2, Osram HBO 200 W) was filtered through a 15%
42
H. NINNEMANN, R. J. SIXASSER and W. L. BUTLER
LMR
I
02MONITOR
w I
U
MC2
LAMP2
Figure 1. Apparatus for simultaneous measurements of changes of absorbance, fluorescence and oxygen content in a sample; ABS: absorption; EB: exciting beam; F : fluorescence; LMR: light mixing rod; LOE: light transmittant oxygen electrode; MB: measuring beam; MC1-MC3: monochromator No. 1-3; PM1, PM2: photomultiplier No. 1, No. 2; S: sample. Insert: With a constant measuring beam and a chopped exciting beam, absorbance and fluorescence are monitored alternately, while the oxygen content is monitored constantly. CuS04 solution (3 cm), a Corning cut-off filter 9782, a Corning broad band blue filter 5562 and through a Bausch and Lomb monochromator (MC 2) set at 436 nm for excitation of flavin fluorescence. The light then passed a light pipe and a light mixing rod reaching the sample with 5 W/m’. The function of the light mixing rod is to “homogenize” the light which could be unevenly distributed across the fibre optics because of an uneven arrangement of the fibres in the light pipe. The actinic light beam could be interrupted with a chopper driven by a synchronous motor with 20rpm, exposing the sample to 1 s of exciting light alternating with 2 s of “dark” (i.e. only measuring light on). Chopping of the actinic light allowed us to measure absorption changes without fluorescence artefacts. The flavin fluorescence excited by this beam passed a light mixing rod, a light pipe, a scanning monochromator (MC 3, Gamma Scientific No. 700-31, San Diego, CA) set at 609 nm and two blocking filters Corning 2418 and 2424. The signal was recognized by a Hamamatsu Gallium Arsenide photomultiplier R 6663 (PM2) and plotted simultaneously with the oxygen content of the sample on a twochannel strip chart recorder. Occurrence of 0; was assayed separately as absorbance increase of nitroblue tetrazolium salt (NBT) at 560 nm or of cytochrome c at 550nm with a Cary 14 or 17 spectrophotometer: the cuvettes contained 50 mM phosphate buffer pH 7.8, 10 mM methionine, cysteine or EDTA as electron .donor, FMN or a flavoprotein and 0.2 mM NBT or 0.02 mM cytochrome c; irradiation with blue or white light generated the superoxide anion which reduced the dye to the blue formazan (Beauchamp and Fridovich, 1971). The absorbance increase could be inhibited with superoxide dismutase (SOD). RESULTS
The influence of tight o n the redox state of insoluble respiratory cytochromes was examined with
freshly purified preparations of complex 1-111 from beef-heart mitochondria. No light-induced absorbance chahges of the cytochromes of complex 1-111 were detected unless soluble flavins were added. In the work reported here complex 1-111 was suspended in the presence of FMN (either 10 or lOOpM) and a suitable electron donor which would support the photoreduction of FMN. EDTA was used as the electron donor in these studies but other compounds such as methionine, dimethylglycine, N-methyl-DL-alanine (Frisell et al., 1959), cystein or glutathione were found to serve nearly as well. Absorption spectra of complex 1-111 in the presence of IOOpM FMN, l O m M EDTA and 25 pM antimycin A measured immediately after an irradiation with blue light (4W/mz a t 460nm) for various periods of time are shown in Fig. 2. Brief periods of irradiation (3-60 s) resulted in the progressive reduction of the two b-type cytochromes in complex 1-111; longer periods of irradiation (90 or 120s) resulted in the reduction of cytochrome c1 as well. The kinetics of the photoreduction of the cytochrome(s) b of complex 1-111, in the presence of 100pM FMN, 10mM EDTA and without or with 25 pM antimycin A, during irradiation with the chopped light source (5 W/m2 at 436 nm) are shown in Fig. 3. The chopped light permitted simultaneous time-course measurements of flavin fluorescence
1
c 530
550
570 nm
530
550
570 nm
Figure 2. Reduced-oxidized difference spectra of complex 1-111 of beef heart mitochondria after irradiation with blue light at room temperature: 1.5 mg CI-I11 protein in 50 mM phosphate buffer pH 7.6 + 100 p M FMN + 10 mM EDTA + 24pM antimycin A. Irradiation time: C-120s; Irradiation with 4 W/mz of blue light (460 nm interference filter, Balzer).
The superoxide anion as electron donor
43
Figure 3. The kinetics of the photoreduction of b-type cytochromes in complex 1-111 in presence of 100 p M FMN simultaneously monitored with the changes of flavin fluorescence and oxygen concentration. Sample 100 p!, containing 0.35 mg CI-111 protein in 50 mM phosphate buffer pH 7.8, 10 mM EDTA, 100pM FMN, $ 2 5 p M antimycin A (AA) +0.5pM superoxide dismutase (SOD). Trace A : change of flavin fluorescence F at 609nm; trace B: change of oxygen content; trace C-E: changes of cytochrome b absorbance at 560nm; C: +EDTA + FMN; D : +EDTA + FMN + AA; E: +EDTA FMN + AA + SOD. Irradiation with monochromatic light (exciting beam) at 436 nm, 5 W/m2. 1 light on, 1 light off.
+
(trace A), oxygen concentration (trace B) and cytochrome b absorbance (traces C-E). Oxygen was consumed during the irradiation in a sequence of reactions which involves the intermediate formation of 0; and the ultimate formation of water (McCord and Fridovich, 1969; Schmidt and Butler, 1976a). As was found previously (Schmidt and Butler, 1976a),the
flavin remained at a high level of oxidation during the irradiation as long as oxygen was present to act as an electron acceptor. When the oxygen was depleted, however, the FMN became reduced in the light as indicated by the decrease of fluorescence. The flavin was reoxidized in the dark by very low levels of oxygen which diffused back into the medium and
44
H. NINNEMANN, R. J. STRASSER and W. L. BUTLER
was fully oxidized after several minutes in the dark. The kinetics of this dark reoxidation of FMN was indicated by the fluorescence excited by the light pulses given at 30s intervals following the second irradiation period (Fig. 3A). In the absence of antimycin A (Fig. 3C), the electrons were fed through to cytochrome c1 rapidly enough that cytochrome(s) b remained oxidized except for a small transient absorbance increase at the onset of light. Cytochrome b became reduced only after cytochrome c1 was completely reduced. Since direct measurements of cytochrome c1 reduction proved to be impossible for technical reasons, this conclusion was inferred from the lag time and the effect of antimycin A: In the presence of antimycin A (Fig. 3D), which blocks electron transport between b- and c-type cytochromes, the cytochrome(s) b started to become reduced immediately at the onset of irradiation. The absorbance measurements in trace D of Fig. 3 indicate that the b-type cytochromes were almost completely reduced at the photostationary state in the presence of oxygen and then became fully reduced when the oxygen was exhausted. The small additional absorbance increase at 560 nm, which occurred when the system became anaerobic, could also be due in part to the reduction of cytochrome cl. It is apparent from the data of Figs. 2 and 3 that the reducing power generated by light in an aerobic system reacted more or less directly at cytochrome(s) b but not at cytochrome cl. When electron transport from cytochrome b to cytochrome c1 was blocked by antimycin A, the cytochrome c1 was reduced only when the system became anaerobic. The photoreduction of cytochrome(s) b is mediated through the generation of 0;. Trace E in Fig. 3 shows the absorbance changes measured at 560 nm when superoxide dismutase was added to the reaction medium. Superoxide dismutase eliminated the 0; and thereby inhibited the photoreduction of the cytochrome. The time course curves for oxygen content and flavin fluorescence were not altered appreciably by the presence of superoxide dismutase. The enzyme rapidly converted 0; to hydrogen peroxide but the rate of oxygen consumption was essentially the same in the absence or presence of superoxide dismutase since the light reaction is rate limiting in both cases. In the presence of superoxide dismutase, however, cytochrome b was not reduced in the light until the system became anaerobic. The reductive power generated by light depends on the intensity of light and the concentration of flavin. The time course measurements of flavin fluorescence, oxygen concentration and cytochrome b reduction during irradiation of complex 1-111 in the presence of 10 pM FMN, 10 mM EDTA and 25 pM antimycin A are shown in Fig. 4. At this lower concentration of flavin, oxygen remained in the medium throughout the 8 min irradiation with 5 W/m2 of 436 nm light and the flavin fluorescence remained high. The rate of production of 0; was lower so that the initial
rate of reduction and the steady-state level of reduction of cytochrome(s) b were lower with the lower concentration of FMN (Fig. 4, trace C); superoxide dismutase blocked the reduction of cytochrome(s) b throughout the irradiation period (Fig. 4, trace D). The lower rate of generation of reducing power with 10 p M FMN, as compared with 100 p M FMN, could have been compensated for by increasing the light intensity. In a previous study (Schmidt and Butler, 1976a), irradiation of a 5 p M solution of flavin with 100 W/m’ of blue light caused the solution to become anaerobic from an air saturated state in about 3 min. It is apparent from the studies with superoxide dismutase that 0; mediates the reduction of cytochrome(s) b. However, the electrons from 0; could be entering the electron transport chain of complex 1-111 any place between the level of NAD and that of cytochrome b. In order to specify the region of interaction more definitively, experiments were also carred out with piericidin (a gift of Dr. T. P. Singer, San Francisco) and rotenone which block electron transport after NADH dehydrogenase. The initial rate of reduction of cytochrome(s)b of complex 1-111 (with 100 p M FMN, 10 mM EDTA and 25 pA4 antimycin A) at the onset of irradiation was essentially as great in the presence of 100pM rotenone or piericidine as it was without those inhibitors (data not shown). Irradiation of purified single complex 111 (containing cytochromes b 5 6 0 , b 5 6 2 and cl) in presence of 100 p M FMN, 10 mM EDTA and 25 pM antimycin A showed similar biphasic reduction kinetics of cytochrome@)b as in complex 1-111, but the 0;-induced reduction level, which could be inhibited by SOD, was comparatively small (Figs. 5A and B). Similar measurements of photoinduced cytochrome reduction in fresh beef heart mitochondria, supplemented with FMN, showed large redox changes of the b-type cytochromes, the kinetics of which were more complicated than those in complex 1-111.
DISCUSSION
Undoubtedly superoxide anion can be formed in mitochondria. Superoxide dismutase (SOD) has been demonstrated in the matrix of mitochondria, e.g. from chicken liver (Weisiger and Fridovich, 1973), rat liver (Panchenko et al., 1975) and in beef heart mitochondria (Keele et al., 1971). McCord ef al. (1971) have shown that the presence of SOD in organisms is strictly associated with their ability to grow under aerobic conditions; thus the presence of this enzyme in a biological system can be taken as indicative of 0; being principally formed there. Chance and Boveris (1972), Loschen et a/. (1971) and others reported that H 2 0 2is produced inside mitochondria. The biological source of 0, in mitochondria is still uncertain. Reduction of O2 to 0, has been shown during reoxidation of reduced flavin (tetraacetylriboflavin; Ballou et ul.. 1969; Massey et a/., 1969) and
The superoxide anion as electron donor
45
Figure 4. The kinetics of the photoreduction of b-type cytochromes in complex 1-111 in presence of 10 p M FMN; simultaneously monitored with the changes of flavin fluorescence and oxygen concentration. Experimental conditions as indicated in legend of Fig. 3 except for .lower FMN concentration (10p M ) . Trace A : flavin fluorescence F at 609 nm; trace B: change of oxygen content; trace C : change of cytochrome b absorbance at 560 in presence of E D T A + FMN (10pM)+ AA; trace D: same in presence of S O D (0.5 pM). Irradiation with 436 nm, 5 W/m2. flavoproteins such as lipoyl dehydrogenase, dihydroorotate dehydrogenase, xanthine oxidase, aldehyde oxidase (Bray et al., 1970; Forman et al., 1975; Komai et al., 1969; Massey et al., 1969; McCord and Fridovich, 1969; Misra and Fridovich, 1972; Rajagopalan and Handler, 1964), or during oxidation of reduced quinones (Boveris et al., 1976; McCord and Fridovich, 1970; Misra and Fridovich, 1972). The most pertinent report in our context is that certain flavincontaining dehydrogenases can generate 0; (Massey et a/., 1969). Loschen et al. (1973) and Loschen (1975) recently published good evidence that HzOzcan be formed
in coupled rat and beef heart mitochondria, involving some component near the second phosphorylation site at the cytochrome b-cl segment of the mitochondrial electron transport chain. Their experiments pointed to the long-wavelength cytochrome b562 (-196°C) being autoxidable and producing 0; in certain metabolic states of the mitochondria. Though HzOz and 0; formation through cytochrome b562 was observed there while 0; consumption by cytochrome@)b is reported in the current paper, the identity of the section or even site concerned in both papers might be more than accidental. In the present paper a well-defined region of the
46
H. NINNEMANN, R. J. STRASSER and W. L. BUTLER
Figure 5. The kinetics of the photoreduction of b-type cytochromes in complex 111. 0.06mg protein in 50 mM phosphate buffer pH 7.6 + 10 mM EDTA + 100 p M FMN + 25 p M antimycin A (AA) & 0.5 p M superoxide dismutase (SOD). A : +EDTA + FMN + AA; B : same in presence of SOD. Irradiation with 436 nm, 5 W/m2. mitochondrial electron transport chain was examined for redox changes that resulted from flavin-mediated photoreduction of cytochrome h via the superoxide anion. Simultaneous measurements of absorbance changes of cytochrome(s) b, flavin fluorescence and of the oxygen content of the mitochondrial system showed that in an aerobic system supplemented with low concentrations of F M N and a flavin-reducing agent, irradiation of freshly prepared NADHcytochrome c reductase (complex I-111), containing two b-type cytochromes (cytochrome b560 and h562) and cytochrome cl, resulted in a selective reduction of the b-type cytochromes but not of cytochrome cl. This reduction of cytochrome b was sensitive to superoxide dismutase. Samples of complex 1-111 supplemented with high concentrations of F M N (100 p M ) became anaerobic within 1 min of irradiation. At that time FMNHz was being formed which reduced unselectively all cytochromes present. The endogenous flavoproteins of complex 1-111 seemed to be unable to mediate the photoreduction of cytochrome(s) b, possibly because the concentration of the non-covalently bound F M N of complex I was too low, or because EDTA may be a poor reductant for these flavoproteins. Experiments with a mitochondrial flavoprotein, lipoyl dehydrogenase from yeast, showed that in vitro this flavoprotein can reduce cytochrome c in the presence of light and cysteine or EDTA as electron donor; the reduction was completely inhibited by SOD
(Ninnemann and Weins, unpublished). Preliminary experiments with yeast submitochondrial particles without supplementing flavin also pointed to flavoprotein-mediated cytochrome c or NBT reduction after irradiation of the mitochondrial fraction. In a series of studies Poff and Butler (1973, 1975), Muiioz and Butler (1975) and Muiioz et ul. (1974) have shown that in v i m a blue light-induced reduction of h-type cytochromes mediated by flavins or flavoproteins is similar to the photoreceptor reaction of various biological “blue light responses”. In a cell-free in vitro system of F M N or riboflavin, cytochrome c and EDTA in phosphate buffer, model reactions for flavin-mediated photoreactions were investigated (Schmidt and Butler, 1976a). The authors proposed 0; as intermediate in the aerobic photostimulated electron transfer from flavin to cytochrome (Muiioz and Butler, 1975; Schmidt and Butler, 1976a; 1976b). Flavoproteins are common constituents in a number of metabolic pathways. Given the general occurrence of flavin-mediated photoreactions, the question arises as to whether light can modify cellular metabolism via flavin-dependent photoreactions. In particular, the photoproduction of 0, could result in light-dependent changes of a number of cellular constituents and pathways. Acknowledgements--We wish to express our special thanks to Dr. Y. Hatefi, Scripps Clinic and Research Foundation, La Jolla, CA. in whose laboratory the enzyme prepar-
The superoxide anion as electron donor ations used in this work were prepared, and who always showed interest in the progress of this work. Thanks are also due to the Deutsche Forschungsgemeinschaft, the
47
Swiss National Fonds and the U.S. Public Health Service for their financial support.
REFERENCES
Ballou, D., G. Palmer and V. Massey (1969) Biochem. Biophys. Res. Commun. 36, 898-904. Beauchamp, C. and I. Fridovich (1971) Anal. Biochem. 44, 276287. Boveris, A., E. Cadenas and A. 0. M. Stoppani (1976) Biochem. J . 156, 435-444. Bray, R. C., F. M. Pick and D. Saumel (1970) Eur. J . Biochem. 15, 352-355. Chance, B. and A. Boveris (1972) Biochem. J . 128, 617-630. Forman, H. J. and J. Kennedy (1975) J . Biol. Chem. 250, 43224326. Frisell, W. R., C. W. Chung and C. G. Mackenzie (1959) J . Biol. Chem. 234, 1297-1302. Natefi, Y., A. G. Harvik and P. Jurtshuk (1961) Biochim. Biophys. Acta 52, 106-118. Hatefi, Y., A. G. Harvik and D. E. Griffiths (1962) J . Biol. Chem. 237, 1681-1685. Keele, B. B., J. M. McCord and I. Fridovich (1971) J . Biol. Chem. 246, 2875-2880. Komai, H., V. Massey and G. Palmer (1969) J . B i d . Chem. 244, 1692-1700. Land, E. J. and A. J. Swallow (1971) Arch. Biochem. Biophys. 145, 365-372. Levander, 0. A., V. C. Morris and D. Higgs (1974) Fed. Proc. 33, 693. Loschen, G. (1975) Dissertation, Tubingen. Loschen, G., L. FlohC and B. Chance (1971) FEBS Lett. 18, 261-264. Loschen, G., A. Azzi and L. Flohe (1973) FEBS Lett. 33, 84-88. Massey, V., S. Strickland, S. G. Mayhew, L. G. Howell, P. C. Engel, R. G. Matthews, M. Schuman and P. A. Sullivan (1969) Biochem. Biophys. Res. Commun. 36, 891-897. McCord, J. M. and I. Fridovich (1968) J . Biol. Chem. 243, 5753-5760. McCord, J. M. and I. Fridovich (1969) J . Biol. Chem. 244, 60494055. McCord, J. M. and I. Fridovich (1970) J . Biol. Chem. 245, 1374-1377. McCord, J. M., B. B. Keele and I. Fridovich (1971) Proc. Natl. Acad. S C I . U.S. 68, 1024-1027. Misra, H. P. and I. Fridovich (1972) J . Biol. Chem. 247, 188-192. Muiioz, V. and W. L. Butler (1975) Plant Physiol. 55, 421-426. Muiioz, V., S. Brody and W. L. Butler (1974) Biochem. Biophys. Res. Cornmun. 58, 322-327. Panchenko, L., 0. S. Brusov, A. M. Gerasimov and T. D. Loktaeva (1975) FEBS Lett. 55, 84-87. Pederson, T. C. and S. D. Aust (1972) Biochem. Biophys. Res. Commun. 48, 789-795. Poff, K. L. and W. L. Butler (1973) Nature (London). 248, 813-816. Poff, K. L. and W. L. Butler (1975) Pfant Physiol. 55, 427-429. Rajagopalan, K. V. and P. Handler (1964) J . Bid. Chem. 239, 2022-2026. Strasser, R. J. (1974) Experientia 30, 320. Schmidt, W. and W. L. Butler (1976a) Photochem. Photobiol. 24, 71-75. Schmidt, W. and W. L. Butler (1976b) Photochem. Photqbiol. 24, 77-80. Weisiger, R. A. and I. Fridovich (1973) J . Biol. Chem. 248, 3582-3592. Zimniermann, R., L. FlohC, U. Weser and H. J. Hartmann (1973) FEBS Lett. 29, 117-120.
Pergamon Press. Printed in Great Britain
THE SUPEROXIDE ANION AS ELECTRON DONOR TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN H. NINNEMANN*, R. J. STRASSER~ and W. L. BUTLERS Department of Biology, University of California at San Diego, La Jolla, CA. 92093, U.S.A. (Received 24 December 1976; accepted 4 February 1977)
Abstract-In isolated respiratory multienzyme complexes of beef heart mitochondria the b-type cytochromes can be photoreduced in presence of flavin via the superoxide anion. 0; does not reduce cytochrome c,. In an anaerobic system, FMNH, formed by irradiation with blue light in presence of EDTA reduces cytochromes b and c l . The possible implication of 0; in the electron transfer from flavin/flavoprotein to cytochrome b in' blue light-controlled biological processes is discussed. INTRODUCTION
well, either by the substances of interest, such as proteins serving as the electron donor for the photoreduction of flavin, or via the hydrogen peroxide generated by the dismutation of 0;.
Superoxide anion is commonly formed in cells or organelles that utilize oxygen. Generally 0; is regarded as being toxic for biological systems either by acting directly as a strong oxidant or reductant, MATERIAL AND METHODS or possibly indirectly by forming H,O, or the exMultienzyme complexes 1-111 (NADH-cytochrome c tremely reactive hydroxyl radical OH'. NADPH- reductase) and 111 (hydroquinone-cytochrome c reductase) dependent peroxidation of microsomal lipids (Peter- were prepared from beef heart mitochondria following the son and Aust, 1972), glutathione-induced swelling of procedure of Hatefi et al. (1961, 1962). The complexes were mitochondria (Levander et al., 1974), glutathione- suspended in TSH buffer at pH 8.0 (0.05 M Tris-CI, 0.66 M sucrose, 1 mM histidine). The superoxide anion 0; was induced lipid peroxidation in isolated inner mito- generated photochemically by irradiating FMN (10 or chondrial membrane fragments (Zimmermann et al., lOOpM) in the presence of EDTA which had been added 1973) are a few of those reactions which seem to in- to the multienzyme complexes. Irradiation was performed with a prefocused Xe lamp volve 0 2 and can be prevented by superoxide dismuEimac Division, VIX-150) in combination with a tase (SOD). There are also reports on 0; serving (Varian, 460 nm interference filter (Balzer) and 2.5 ern CuSO, soluas electron donor to a solution of ferri-cytochrome tion (15%) resulting in 4 W/mz. Within seconds after the c (McCord and Fridovich, 1968; 1969; Land and irradiation, absorption spectra were measured at room Swallow, 1971). This reaction has been used as a temperature with a Cary 14 spectrophotometer (tungsten simple assay for the presence of the superoxide anion. lamp, EM1 9558C phototube and logarithmic photometer) on line with a Digital PDP/8 computer with a 12-bit We wish to report experiments which show that analog-to-digital converter. The single beam absorption irradiation of mitochondria1 multienzyme complexes spectra were stored in the memory core of the computer 1-111 and 111 of beef heart mitochondria supple- and displayed as difference spectra between sample and mented with soluble flavins and an electron donor reference. Simultaneous measurements of absorption changes of to the flavin resulted in a steady uptake of oxygen cytochrome b at 560 nm (AA 560), of changes in flavin fluat a rate which depended on the intensity of light orescence (measured at 609 nm, AF 609) and of the oxygen and the concentration of flavin. The 0; formed in content of the enzyme system were performed with an optithese reactions served as a selective electron donor cal set-up constructed in this laboratory (R. Strasser) to the b-type cytochromes of the respiratory electron (Fig. 1). The measuring beam (MB) from a tungsten iodine lamp (lamp 1) passed through two Corning cut-off transport chain. Irradiating the flavin-supplemented filters 3486, an interference filter S1 with T,,,= 560nm multienzyme complexes under anaerobic conditions and through a Bausch and Lomb monochromator (MC 1) led to the photoreduction of the flavin and, under set at 560 nm for measuring cytochrome b absorption these conditions, the reduced flavin reacted directly changes. It then passed a light pipe (Schott, Mainz, W. Germany), a light mixing rod (LMR) and an inverted lightto reduce the cytochromes b in suspension. In addi- transmittant oxygen electrode (Clark type, LOE; Strasser, tion to these flavin-mediated photoreductive reac- 1974). The sample S (loo@, 1 mm thickness) was placed tions, photooFidative reactions may be generated as between this 0,-electrode and a second light mixing rod *On leave of absence from the University of Tubingen, W. Germany. Supported by the Deutsche Forschungsgemeinschaft by grants No. Ni 119/4 and 5. Present address: Institut fur Chemische Pflanzenphysiologie, CorrensstraBe 41, 7400 Tubingen, W. Germany. ?Supported by the Swiss National Fonds. $Supported by US. Public Health Service grant GM 20648. 41
(LMR). Here the measuring light was picked up behind the sample, guided through a filter combination of Corning cut off filter 3486 and Balzer interference filters 557 and 562nm to a Hamamatsu phototube R818 (PM1). The absorbance changes were plotted on a Hewlett Packard 7074A recorder where off-setting and proper amplification allowed a full scale sensitivity of 0.008 A per 20 cm. The actinic light from a high intensity mercury lamp (lamp 2, Osram HBO 200 W) was filtered through a 15%
42
H. NINNEMANN, R. J. SIXASSER and W. L. BUTLER
LMR
I
02MONITOR
w I
U
MC2
LAMP2
Figure 1. Apparatus for simultaneous measurements of changes of absorbance, fluorescence and oxygen content in a sample; ABS: absorption; EB: exciting beam; F : fluorescence; LMR: light mixing rod; LOE: light transmittant oxygen electrode; MB: measuring beam; MC1-MC3: monochromator No. 1-3; PM1, PM2: photomultiplier No. 1, No. 2; S: sample. Insert: With a constant measuring beam and a chopped exciting beam, absorbance and fluorescence are monitored alternately, while the oxygen content is monitored constantly. CuS04 solution (3 cm), a Corning cut-off filter 9782, a Corning broad band blue filter 5562 and through a Bausch and Lomb monochromator (MC 2) set at 436 nm for excitation of flavin fluorescence. The light then passed a light pipe and a light mixing rod reaching the sample with 5 W/m’. The function of the light mixing rod is to “homogenize” the light which could be unevenly distributed across the fibre optics because of an uneven arrangement of the fibres in the light pipe. The actinic light beam could be interrupted with a chopper driven by a synchronous motor with 20rpm, exposing the sample to 1 s of exciting light alternating with 2 s of “dark” (i.e. only measuring light on). Chopping of the actinic light allowed us to measure absorption changes without fluorescence artefacts. The flavin fluorescence excited by this beam passed a light mixing rod, a light pipe, a scanning monochromator (MC 3, Gamma Scientific No. 700-31, San Diego, CA) set at 609 nm and two blocking filters Corning 2418 and 2424. The signal was recognized by a Hamamatsu Gallium Arsenide photomultiplier R 6663 (PM2) and plotted simultaneously with the oxygen content of the sample on a twochannel strip chart recorder. Occurrence of 0; was assayed separately as absorbance increase of nitroblue tetrazolium salt (NBT) at 560 nm or of cytochrome c at 550nm with a Cary 14 or 17 spectrophotometer: the cuvettes contained 50 mM phosphate buffer pH 7.8, 10 mM methionine, cysteine or EDTA as electron .donor, FMN or a flavoprotein and 0.2 mM NBT or 0.02 mM cytochrome c; irradiation with blue or white light generated the superoxide anion which reduced the dye to the blue formazan (Beauchamp and Fridovich, 1971). The absorbance increase could be inhibited with superoxide dismutase (SOD). RESULTS
The influence of tight o n the redox state of insoluble respiratory cytochromes was examined with
freshly purified preparations of complex 1-111 from beef-heart mitochondria. No light-induced absorbance chahges of the cytochromes of complex 1-111 were detected unless soluble flavins were added. In the work reported here complex 1-111 was suspended in the presence of FMN (either 10 or lOOpM) and a suitable electron donor which would support the photoreduction of FMN. EDTA was used as the electron donor in these studies but other compounds such as methionine, dimethylglycine, N-methyl-DL-alanine (Frisell et al., 1959), cystein or glutathione were found to serve nearly as well. Absorption spectra of complex 1-111 in the presence of IOOpM FMN, l O m M EDTA and 25 pM antimycin A measured immediately after an irradiation with blue light (4W/mz a t 460nm) for various periods of time are shown in Fig. 2. Brief periods of irradiation (3-60 s) resulted in the progressive reduction of the two b-type cytochromes in complex 1-111; longer periods of irradiation (90 or 120s) resulted in the reduction of cytochrome c1 as well. The kinetics of the photoreduction of the cytochrome(s) b of complex 1-111, in the presence of 100pM FMN, 10mM EDTA and without or with 25 pM antimycin A, during irradiation with the chopped light source (5 W/m2 at 436 nm) are shown in Fig. 3. The chopped light permitted simultaneous time-course measurements of flavin fluorescence
1
c 530
550
570 nm
530
550
570 nm
Figure 2. Reduced-oxidized difference spectra of complex 1-111 of beef heart mitochondria after irradiation with blue light at room temperature: 1.5 mg CI-I11 protein in 50 mM phosphate buffer pH 7.6 + 100 p M FMN + 10 mM EDTA + 24pM antimycin A. Irradiation time: C-120s; Irradiation with 4 W/mz of blue light (460 nm interference filter, Balzer).
The superoxide anion as electron donor
43
Figure 3. The kinetics of the photoreduction of b-type cytochromes in complex 1-111 in presence of 100 p M FMN simultaneously monitored with the changes of flavin fluorescence and oxygen concentration. Sample 100 p!, containing 0.35 mg CI-111 protein in 50 mM phosphate buffer pH 7.8, 10 mM EDTA, 100pM FMN, $ 2 5 p M antimycin A (AA) +0.5pM superoxide dismutase (SOD). Trace A : change of flavin fluorescence F at 609nm; trace B: change of oxygen content; trace C-E: changes of cytochrome b absorbance at 560nm; C: +EDTA + FMN; D : +EDTA + FMN + AA; E: +EDTA FMN + AA + SOD. Irradiation with monochromatic light (exciting beam) at 436 nm, 5 W/m2. 1 light on, 1 light off.
+
(trace A), oxygen concentration (trace B) and cytochrome b absorbance (traces C-E). Oxygen was consumed during the irradiation in a sequence of reactions which involves the intermediate formation of 0; and the ultimate formation of water (McCord and Fridovich, 1969; Schmidt and Butler, 1976a). As was found previously (Schmidt and Butler, 1976a),the
flavin remained at a high level of oxidation during the irradiation as long as oxygen was present to act as an electron acceptor. When the oxygen was depleted, however, the FMN became reduced in the light as indicated by the decrease of fluorescence. The flavin was reoxidized in the dark by very low levels of oxygen which diffused back into the medium and
44
H. NINNEMANN, R. J. STRASSER and W. L. BUTLER
was fully oxidized after several minutes in the dark. The kinetics of this dark reoxidation of FMN was indicated by the fluorescence excited by the light pulses given at 30s intervals following the second irradiation period (Fig. 3A). In the absence of antimycin A (Fig. 3C), the electrons were fed through to cytochrome c1 rapidly enough that cytochrome(s) b remained oxidized except for a small transient absorbance increase at the onset of light. Cytochrome b became reduced only after cytochrome c1 was completely reduced. Since direct measurements of cytochrome c1 reduction proved to be impossible for technical reasons, this conclusion was inferred from the lag time and the effect of antimycin A: In the presence of antimycin A (Fig. 3D), which blocks electron transport between b- and c-type cytochromes, the cytochrome(s) b started to become reduced immediately at the onset of irradiation. The absorbance measurements in trace D of Fig. 3 indicate that the b-type cytochromes were almost completely reduced at the photostationary state in the presence of oxygen and then became fully reduced when the oxygen was exhausted. The small additional absorbance increase at 560 nm, which occurred when the system became anaerobic, could also be due in part to the reduction of cytochrome cl. It is apparent from the data of Figs. 2 and 3 that the reducing power generated by light in an aerobic system reacted more or less directly at cytochrome(s) b but not at cytochrome cl. When electron transport from cytochrome b to cytochrome c1 was blocked by antimycin A, the cytochrome c1 was reduced only when the system became anaerobic. The photoreduction of cytochrome(s) b is mediated through the generation of 0;. Trace E in Fig. 3 shows the absorbance changes measured at 560 nm when superoxide dismutase was added to the reaction medium. Superoxide dismutase eliminated the 0; and thereby inhibited the photoreduction of the cytochrome. The time course curves for oxygen content and flavin fluorescence were not altered appreciably by the presence of superoxide dismutase. The enzyme rapidly converted 0; to hydrogen peroxide but the rate of oxygen consumption was essentially the same in the absence or presence of superoxide dismutase since the light reaction is rate limiting in both cases. In the presence of superoxide dismutase, however, cytochrome b was not reduced in the light until the system became anaerobic. The reductive power generated by light depends on the intensity of light and the concentration of flavin. The time course measurements of flavin fluorescence, oxygen concentration and cytochrome b reduction during irradiation of complex 1-111 in the presence of 10 pM FMN, 10 mM EDTA and 25 pM antimycin A are shown in Fig. 4. At this lower concentration of flavin, oxygen remained in the medium throughout the 8 min irradiation with 5 W/m2 of 436 nm light and the flavin fluorescence remained high. The rate of production of 0; was lower so that the initial
rate of reduction and the steady-state level of reduction of cytochrome(s) b were lower with the lower concentration of FMN (Fig. 4, trace C); superoxide dismutase blocked the reduction of cytochrome(s) b throughout the irradiation period (Fig. 4, trace D). The lower rate of generation of reducing power with 10 p M FMN, as compared with 100 p M FMN, could have been compensated for by increasing the light intensity. In a previous study (Schmidt and Butler, 1976a), irradiation of a 5 p M solution of flavin with 100 W/m’ of blue light caused the solution to become anaerobic from an air saturated state in about 3 min. It is apparent from the studies with superoxide dismutase that 0; mediates the reduction of cytochrome(s) b. However, the electrons from 0; could be entering the electron transport chain of complex 1-111 any place between the level of NAD and that of cytochrome b. In order to specify the region of interaction more definitively, experiments were also carred out with piericidin (a gift of Dr. T. P. Singer, San Francisco) and rotenone which block electron transport after NADH dehydrogenase. The initial rate of reduction of cytochrome(s)b of complex 1-111 (with 100 p M FMN, 10 mM EDTA and 25 pA4 antimycin A) at the onset of irradiation was essentially as great in the presence of 100pM rotenone or piericidine as it was without those inhibitors (data not shown). Irradiation of purified single complex 111 (containing cytochromes b 5 6 0 , b 5 6 2 and cl) in presence of 100 p M FMN, 10 mM EDTA and 25 pM antimycin A showed similar biphasic reduction kinetics of cytochrome@)b as in complex 1-111, but the 0;-induced reduction level, which could be inhibited by SOD, was comparatively small (Figs. 5A and B). Similar measurements of photoinduced cytochrome reduction in fresh beef heart mitochondria, supplemented with FMN, showed large redox changes of the b-type cytochromes, the kinetics of which were more complicated than those in complex 1-111.
DISCUSSION
Undoubtedly superoxide anion can be formed in mitochondria. Superoxide dismutase (SOD) has been demonstrated in the matrix of mitochondria, e.g. from chicken liver (Weisiger and Fridovich, 1973), rat liver (Panchenko et al., 1975) and in beef heart mitochondria (Keele et al., 1971). McCord ef al. (1971) have shown that the presence of SOD in organisms is strictly associated with their ability to grow under aerobic conditions; thus the presence of this enzyme in a biological system can be taken as indicative of 0; being principally formed there. Chance and Boveris (1972), Loschen et a/. (1971) and others reported that H 2 0 2is produced inside mitochondria. The biological source of 0, in mitochondria is still uncertain. Reduction of O2 to 0, has been shown during reoxidation of reduced flavin (tetraacetylriboflavin; Ballou et ul.. 1969; Massey et a/., 1969) and
The superoxide anion as electron donor
45
Figure 4. The kinetics of the photoreduction of b-type cytochromes in complex 1-111 in presence of 10 p M FMN; simultaneously monitored with the changes of flavin fluorescence and oxygen concentration. Experimental conditions as indicated in legend of Fig. 3 except for .lower FMN concentration (10p M ) . Trace A : flavin fluorescence F at 609 nm; trace B: change of oxygen content; trace C : change of cytochrome b absorbance at 560 in presence of E D T A + FMN (10pM)+ AA; trace D: same in presence of S O D (0.5 pM). Irradiation with 436 nm, 5 W/m2. flavoproteins such as lipoyl dehydrogenase, dihydroorotate dehydrogenase, xanthine oxidase, aldehyde oxidase (Bray et al., 1970; Forman et al., 1975; Komai et al., 1969; Massey et al., 1969; McCord and Fridovich, 1969; Misra and Fridovich, 1972; Rajagopalan and Handler, 1964), or during oxidation of reduced quinones (Boveris et al., 1976; McCord and Fridovich, 1970; Misra and Fridovich, 1972). The most pertinent report in our context is that certain flavincontaining dehydrogenases can generate 0; (Massey et a/., 1969). Loschen et al. (1973) and Loschen (1975) recently published good evidence that HzOzcan be formed
in coupled rat and beef heart mitochondria, involving some component near the second phosphorylation site at the cytochrome b-cl segment of the mitochondrial electron transport chain. Their experiments pointed to the long-wavelength cytochrome b562 (-196°C) being autoxidable and producing 0; in certain metabolic states of the mitochondria. Though HzOz and 0; formation through cytochrome b562 was observed there while 0; consumption by cytochrome@)b is reported in the current paper, the identity of the section or even site concerned in both papers might be more than accidental. In the present paper a well-defined region of the
46
H. NINNEMANN, R. J. STRASSER and W. L. BUTLER
Figure 5. The kinetics of the photoreduction of b-type cytochromes in complex 111. 0.06mg protein in 50 mM phosphate buffer pH 7.6 + 10 mM EDTA + 100 p M FMN + 25 p M antimycin A (AA) & 0.5 p M superoxide dismutase (SOD). A : +EDTA + FMN + AA; B : same in presence of SOD. Irradiation with 436 nm, 5 W/m2. mitochondrial electron transport chain was examined for redox changes that resulted from flavin-mediated photoreduction of cytochrome h via the superoxide anion. Simultaneous measurements of absorbance changes of cytochrome(s) b, flavin fluorescence and of the oxygen content of the mitochondrial system showed that in an aerobic system supplemented with low concentrations of F M N and a flavin-reducing agent, irradiation of freshly prepared NADHcytochrome c reductase (complex I-111), containing two b-type cytochromes (cytochrome b560 and h562) and cytochrome cl, resulted in a selective reduction of the b-type cytochromes but not of cytochrome cl. This reduction of cytochrome b was sensitive to superoxide dismutase. Samples of complex 1-111 supplemented with high concentrations of F M N (100 p M ) became anaerobic within 1 min of irradiation. At that time FMNHz was being formed which reduced unselectively all cytochromes present. The endogenous flavoproteins of complex 1-111 seemed to be unable to mediate the photoreduction of cytochrome(s) b, possibly because the concentration of the non-covalently bound F M N of complex I was too low, or because EDTA may be a poor reductant for these flavoproteins. Experiments with a mitochondrial flavoprotein, lipoyl dehydrogenase from yeast, showed that in vitro this flavoprotein can reduce cytochrome c in the presence of light and cysteine or EDTA as electron donor; the reduction was completely inhibited by SOD
(Ninnemann and Weins, unpublished). Preliminary experiments with yeast submitochondrial particles without supplementing flavin also pointed to flavoprotein-mediated cytochrome c or NBT reduction after irradiation of the mitochondrial fraction. In a series of studies Poff and Butler (1973, 1975), Muiioz and Butler (1975) and Muiioz et ul. (1974) have shown that in v i m a blue light-induced reduction of h-type cytochromes mediated by flavins or flavoproteins is similar to the photoreceptor reaction of various biological “blue light responses”. In a cell-free in vitro system of F M N or riboflavin, cytochrome c and EDTA in phosphate buffer, model reactions for flavin-mediated photoreactions were investigated (Schmidt and Butler, 1976a). The authors proposed 0; as intermediate in the aerobic photostimulated electron transfer from flavin to cytochrome (Muiioz and Butler, 1975; Schmidt and Butler, 1976a; 1976b). Flavoproteins are common constituents in a number of metabolic pathways. Given the general occurrence of flavin-mediated photoreactions, the question arises as to whether light can modify cellular metabolism via flavin-dependent photoreactions. In particular, the photoproduction of 0, could result in light-dependent changes of a number of cellular constituents and pathways. Acknowledgements--We wish to express our special thanks to Dr. Y. Hatefi, Scripps Clinic and Research Foundation, La Jolla, CA. in whose laboratory the enzyme prepar-
The superoxide anion as electron donor ations used in this work were prepared, and who always showed interest in the progress of this work. Thanks are also due to the Deutsche Forschungsgemeinschaft, the
47
Swiss National Fonds and the U.S. Public Health Service for their financial support.
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