ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 288, No. 1, July, pp. 112-117, 1991
Cytochrome c-Catalyzed Oxidation of Organic Molecules by Hydrogen Peroxide Rafael Radi,’ Leonor Thomson,
Homer0 Rubbo, and Eugenio Prodanov’
Department of Biochemistry, Faculty of Medicine, University Au. Gral. Flares 2125, Montevideo, Uruguay CP 11800
Received December 14, 1990, and in revised form February
of the Republic,
26, 1991
Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (HzO,), including 2-2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the HzOz-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with HzOz increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (0;) and hydroxyl radical (‘OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H202 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H,Oz-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of HzOz with an oxidized luminol molecule, either luminol radical or luminol diazoquinone. 0 issi Academic PESS, IN.
ported that cytochrome c participates in the hydroxylation of 4-nitrophenol (2) and the oxidation of 2-keto-4-thiomethyl butyric acid (3) in the presence of HzOz. On the other hand, cytochrome c is degraded by H202 and hydroperoxides, with irreversible spectral changes and production of spontaneous chemiluminescence (4). During the interaction of cytochrome c with H202, hydroperoxides, and reducing substrates, the mechanism of iron involvement and the participating active species have not been well defined. Cytochrome c2+ can be reoxidized to cytochrome c3+ by H202, giving rise possibly to ‘OH (5, 6). When cytochrome c3+ reacts with HzOz formation of a bound hydroxyl radical (Fe3+--‘OH) by a site-specific reaction at the heme was proposed (4). We have previously shown that ferricytochrome c increases luminol chemiluminescence produced by the xanthine oxidase/hypoxanthine/OZ system (7). Because cytochrome c3+ scavenges 0, (8) and luminol chemiluminescence is usually dependent on 0; (7, 9, lo), the light emission increase was unexpected and suggested a more complex role of cytochrome c. In this work we studied the reactions of cytochrome c with Hz02 and the secondary oxidation of organic molecules. Herein we report a kinetic analysis of this catalytic action of cytochrome c and concurrently communicate an alternative mechanism of luminol chemiexcitation by hemeproteins. These data suggest that cytochrome c might play a role in mediating or enhancing cytotoxic effects of HzOz. MATERIALS
AND
METHODS
While the peroxidatic activity of several hemoproteins and hematin is well understood (l), the mechanism and kinetics of cytochrome c-catalyzed oxidation of substrates by HzOz has not been explored in detail. It has been re-
Cytochrome c (types III and VI), luminol (5amino-2,3-dihydro-1,4phtalazinedione), lucigenin (his-N-methylacridinium nitrate), 2-2’-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS): 4-aminoantipyrine (4.AP), diethylene triaminopentaacetic acid (DTPA), 2thiobarbituric
i Current address: Departments of Anesthesiology and Biochemistry, The University of Alabama at Birmingham, 619 19th St. South, 941 THT, Birmingham, Alabama 35233. ’ To whom correspondence should be addressed.
3 Abbreviations used: ABTS, 2-2’.azino-bis (3-ethylbenzthiazoline-6sulfonic acid); 4-AP, 4-amino-antipyrine; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid; HRP, horseradish peroxidase.
112
0003.9861/91$3.00 All
Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
CYTOCHROME
c AND
acid and mannitol were obtained from Sigma. Hydrogen peroxide was obtained from Baker; uric acid and sodium dithionite were from Fisher. All other reagents used were of analytical grade. Cytochrome c was reduced by sodium dithionite and purified by chromatography on Sephadex G-25 using 10 mM potassium phosphate, pH 7.0, as the elution buffer. In some experiments cytochrome c3’ was also purified on Sephadex G-25 in order to remove potential hemopeptide residues with microperoxidase activity (6). No differences in the reactions catalyzed by cytochrome c were observed when compared to untreated cytochrome c3+. Spectrophotometric measurements were performed in a Shimadzu uv 160 spectrophotometer. Oxidation rates of 4-AP (6~ = 6.4 X 10” Mu ‘. cm-‘, Ref. (2)) and ABTS (tM = 3.6 X lo4 M-’ . cm- ‘, Ref. (10)) were followed at 505 and 420 nm, respectively. Hydroxyl radical formation was studied by deoxyribose degradation to 2.thiobarbituric acid reactive material (11). Chemiluminescent reactions were carried out under continuous oxygen bubbling as described before (7, 12). Nitrogen replaced oxygen in some cases for anaerobic studies. A 0.1 M pyrophosphate buffer, pH 8.3, was used for chemiluminescent reactions. For other assays, buffer systems were chosen which were effective at a particular pH range; to rule out remote buffer effects on catalytic activity, reaction rates were determined at each pH with two different buffer systems. None of the buffers reported in this manuscript significantly inhibited cyt,ochrome c/H,O,-induced ABTS and 4-AP oxidation. Each set of experiments reported herein was repeated a minimum of three times with similar results. Data represent the mean of two different analyses from a representative experiment.
RESULTS
Oxidation of ABTS and 4-AP by H,O, in the Presence of Cytochrome c ABTS and 4-AP were oxidized by H202 in the presence of cytochrome eni. Reactions obeyed Michaelis-Menten kinetics for H202, having K, values of 65 mM at pH 6.0 to 7.5 at saturating concentrations of the reductants, as determined by a Lineweaver-Burk plot (Fig. 1). This K, represents a binding energy of approximately 4.1 kcal/ mol for the interaction cytochrome c-HzOz, calculated as unitary free energy (13). Maximal dependence of velocity on ABTS concentration was found in a very narrow range correfrom 1 to 6 yM and the substrate concentration sponding to half-maximal reaction velocity was within the micromolar concentration range. The reaction velocity remained nearly constant when the ABTS concentration was higher than 10 PM. Second-order constants were 3 X 10’ [H202]. mini’ for ABTS and 4.1 X lo5 [ABTS] . min * for H,O,. Horseradish peroxidase (HRP) oxidized ABTS and 4-AP at lo4 to lo5 faster rates than equimolar cytochrome c”+, at pH 6.0. Oxidation rates were directly proportional to cytochrome c3+ concentration from 0.75 to 30 WM. When cytochrome es+ was used there was a lag phase before the oxidation of ABTS or 4-AP started. This lag phase corresponded to the time required to fully oxidize cytochrome c2+to the ferric state in the presence of HZ02, monitored spectroscopically as the disappearance of the 550-nm band (characteristic of cytochrome c2+, tM = 2,l X 10’ Mm’ * cm-‘; Ref. (8)). The second-order rate constant
HYDROGEN
113
PEROXIDE
c2+ reoxidation by HzOz was 125 cytochrome emin’, consistent with other observations (6). Cyanide inhibited the oxidation of ABTS and 4-AP by cytochrome c3+ and HaOz. Oxidation rates of ABTS in the presence of 6 PM cytochrome c3+ plus 0.73 or 4.4 mM H202 at pH 5.6 were inhibited by 60 and 45%, respectively, using 0.17 mM cyanide. The pH profile of the catalytic activity of cytochrome c is represented in Fig. 2. This activity was higher in the acidic pH range, with a maximum at pH 3.6-3.8. There was no activity toward ABTS and 4-AP at pH > 9.5. of
M-r
Catalytic Activity of Cytochrome c and Its Relation with Spectral Changes Brief preincubation of cytochrome c3+ with H,Oz increased catalytic activity; longer pre-incubation led to a progressive decrease (Fig. 3). At the same time, the spectra of cytochrome c showed a continuous exponential decrease of the Soret band (408-410 nm; not shown). Degradation of cytochrome c by H202 showed a second-order rate constant (h’) of 14.2 [H202] emin’ at 18”C, pH 6.0 (Table I). Diethylenetriaminepentaacetic acid, SOD, and mannitol added to the preincubation medium did not alter catalytic activity or the spectral changes. Tryptophan (5 mM) inhibited cytochrome c degradation and oxidation rates by approximately 15%, with uric acid having an even more potent inhibition. Uric acid influence on catalytic activity and cytochrome c degradation depended heavily on uric acid/H20Z molar ratios (Fig. 3, Table I). When uric acid:HpOl molar ratios were low (l:lO), no effects of uric acid were observed. With ratios around l:l, cytochrome c activation was delayed (Fig. 3) as well as inactivation (Fig. 3) and degradation (Table I). Uric acid in
-0.02
0.10
0.04 [H,OJ-’
0.16
hM)-’
FIG. 1. Michaelis constant (K,) determination for HzO,. Different concentrations of H,O, (6.7 to 80 mM) were incubated in the presence of 0.6 pM cytochrome c3+ and 20 mM ABTS in 0.067 M potassium phosphate, pH 7.0, at 18°C. Initial reaction rates were obtained by spectrophotometric measurement of ABTS oxidation.
1 14
RADI
ET AL. TABLE
0.05
Influence
of Uric
0.04
?
Acid and H,Os on Cytochrome Degradation Rates
[Uric acidJ/[H,Os]
E 0.03
t E
I
k’([H,O,]
0 0.1 0.4 1.0 5.0
>" 0.02
0.01
3
5
7
9
11
PH FIG. 2. Influence of pH on catalytic activity. Assay conditions were: 6 pM cytochrome c3+, 0.6 mM H202 and 1.3 mM ABTS at different pH, 18’C. The buffer systems used were 0.05 M phtalate-HCl, pH 2.0 to 5.0; 0.05 M citrate-phosphate, pH 5.4 to 7.0; 0.05 M pyrophosphate, pH 8.0 and 8.5; 0.05 M borate, pH 9.0 to 10.0.
c3+
.min-i)
14.2 14.2 14.2 11.1 4.7
Note. Cytochrome c 3t (67 FM) was incubated with 1.9 mM H202 and different uric acid concentrations in 0.067 M potassium phosphate, pH 6.0, at 18°C. At different time points aliquots were taken and diluted to a final concentration of 6.7 @M cytochrome c for spectroscopic measurements. Spectral changes were followed by the disappearance of Soret band absorbance at 408 nm. Data represents X (n = 2) from a representative experiment which had a maximum variation of 0.02 [H,O,] mini between replicates.
4). There was a lag phase and a lower intensity peak of Chemilucytochrome c2+-induced chemiluminescence. excess over H202 (5:l) completely suppressed catalytic minescent yields and lag phases were dependent on HzOz activity and substantially inhibited cytochrome c degraconcentration (Fig. 5). Lag phases of photoemission were dation (Table I). shortened and even disappeared if cytochrome c2+ was No 2-thiobarbituric acid reactive material was detected after deoxyribose was incubated with cytochrome c3+ preincubated with HzOz. The dependence of chemiluminescence on luminol concentration is shown in Fig. 6. and H202. Initial velocities of chemiluminescence increase (ucL) were measured as the initial slope of light intensity versus time. Chemiluminescence Produced by a Cytochrome Data were fitted to a Lineweaver-Burk plot with an apc-H202-Luminol System parent K,,, of 84 PM. Both ferro and ferric forms of cytochrome c were caHorseradish peroxidase concentrations equimolar with pable of producing luminol chemiluminescence in the cytochrome c induced a very strong luminol-dependent presence of H20z, but with different time courses (Fig. light emission, in the order of lo3 bigger than that of cy-
1200
900
.L 0
10 preincubatlon
20 time
30
7--
0
a
10
20
30
(mid
FIG. 3. Influence of preincubation with H,O, on catalytic activity. Cytochrome c3+ (83 pM) was pr eincubated with 0.6 mM H202 in the absence (0) or presence (0) of 0.4 mM uric acid in 0.067 M potassium phosphate at 18°C. At different times aliquots were added to 27 mM 4AP. Final concentrations of cytochrome c and H,Oz after dilution were 9 pM and 0.065 mM, respectively. Initial reaction rates were obtained by following 4-AP oxidation spectrophotometrically.
time
(min)
FIG. 4. Time course of cyt c/HsO*-induced luminol chemiluminescence. Reagent concentrations were: cytochrome c3+ (A) or cytochrome c*+ (0) 25 pM and HRP (0) 25 nM; lumino10.67 mM and HzOs, 0.6 mM in 0.1 M pyrophosphate pH at 18°C. Reactions were initiated by addition of the cytochrome. Light intensity was measured in photometer scale units.
CYTOCHROME
c AND
HYDROGEN
115
PEROXIDE
4
600
3 400
L
,x 2 a, .-E E .P 200
0 0 I
5
IO
15
20
OL -20 25
hnird-’
time (min)
FIG. 5. Time course of luminol chemiluminescence as a function of H,Os concentration. Concentrations of the reagents were: cytochrome I?, 30 PM; luminol, 0.67 mM, and H202, 0.15 mM (e), 0.30 mM (W), 0.45 mM (O), 0.60 mM (A.). Reactions were conducted in 0.10 M pyrophosphate, pH 8.3, at 18°C. Light intensity was measured in photometer scale units.
tochrome c, which decayed very rapidly (~15 s) due to HzOz depletion. When HRP was diluted lo3 times, chemiluminescence yield was similar to that induced by cytochrome c, without a lag phase (Fig. 4). Luminol chemiluminescence induced by cytochrome c/H202 was inhibited instantaneously and completely by 1 mM uric acid, almost completely by 1 mM tryptophan, with SOD and mannitol having no effect (Table II). No luminol chemiluminescence was observed when uric acid was present at the beginning of the reaction. The chemiluminescent probe, lucigenin, gave a very weak light emission with a maximal intensity of about 0.2% of that of luminol. No dependence upon oxygen concentration was found for catalytic activity toward reducing substrates, including luminol (not shown).
knM)-’
FIG. 6. Influence of luminol concentration on chemiluminescence. The reaction mixtures contained 30 PM cytochrome c3+, 0.6 mM H202 and different luminol concentrations (0.02 to 0.66 mM) in 0.1 M pyrophosphate, pH 8.3, at 18’C. Rates of luminol oxidation were determined as the initial slope of light intensity (photometer scale units) versus time (seconds).
high K,,, for H202 indicate that the donor molecules bind to cytochrome c at sites other than the ferric heme iron. Peroxidases bind their electron donor substrates in hydrophobic clefts in close vicinity to the heme moiety (15, 16). Binding to a cytochrome c hydrophobic cleft (involving multiple noncovalent hydrophobic interactions) is in agreement with high affinities found for reducing substrates. An interesting problem concerns the mechanism of substrate oxidation. Ferrous iron was not involved, because with ferrocytochrome c a lag phase preceded the beginning of the peroxidatic activity, corresponding to the oxidation of the ferrous to the ferric form of cytochrome c. With ferricytochrome c, no lag time was observed, although there was often an acceleration before
DISCUSSION
Our results support a catalytic mechanism for the oxidation of organic molecules by cytochrome c in the presence of Hz02. Data are consistent with a Michaelis-Menten kinetics for HzOz having a K, of approximately 65 mM (Fig. 1). This K, reflects a very weak affinity for HzOz, consistent with a nonionic and noncovalent bond of approximately 4.1 kcal/mol between HzOz and cytochrome c. Inhibition of ABTS and 4-AP oxidation by cyanide supports the idea that catalysis by cytochrome c involves the sixth ligand position of the iron in binding HA (4, 14). There is a low specificity of cytochrome c toward electron donors as demonstrated herein and elsewhere (2, 3) but the kinetic data supports a saturation behavior. The reductants appeared to bind to cytochrome c as shown by the K, value for luminol and the Ko.5for ABTS. The low Km found for the electron donors in comparison to the
TABLE
II
Effect of Free Radical Scavengers on Cytochrome c3+Plus H,Oz-Induced Luminol Chemiluminescence Reagent None Urate Tryptophan Mannitol SOD
Light intensity 120 0 15 120 120
% Inhibition
100 87 0 0
Note. Cytochrome c3+ (10 PM) was incubated with 0.84 mM H,O, and 0.067 mM luminol in 0.1 M pyrophosphate, pH 8.3,18”C. A steady-state chemiluminescence of 120 photometer scale units was obtained by 20 min. At t = 23 min, 1 mM mannitol, 1 mM tryptophan, 1 mM urate, or 1 PM SOD was added. Data reports inhibition of photoemission immediately after addition of scavengers; in all cases percentage inhibition remained constant during a 15-min observation period.
116
RADI
attaining a steady-state. The fact that catalytic activity increased after a short preincubation of ferricytochrome c with hydrogen peroxide (Fig. 3) strongly suggests that ferricytochrome c was not the actual active form but a precursor. Our experiments give no direct evidence for the oxidation state of iron, but the lack of effect of SOD makes it very unlikely to be a catalyzed Haber-Weiss type mechanism. Lag times obtained with ferrocytochrome c, insensitivity to mannitol, and absence of deoxyribose degradation exclude a Fenton-like reaction producing ‘OH radical. Therefore, the catalytically active form of cytochrome c must bear iron in a more oxidized state, as in compound I of peroxidases where a ferry1 (Fe4+=0) ion is bound to a porphyrin cation radical. Ferryl ion has similar reactivity and is kinetically indistinguishable from ‘OH (17, 18). Formation of the ferry1 species is in good agreement with the inhibitory effects of uric acid in either substrate oxidation or cytochrome c degradation since uric acid is an excellent scavenger of oxo-heme oxidants (19), competing for the oxidant with the substrates or the sensitive groups of the protein. ln the case of cytochrome c, however, this high oxidation state protein species must be very unstable and would be easily degraded by reaction with HzOz or by internal rearrangement of the oxidized molecule. The fact that maximum activity is reached after a definite time interval (Fig. 3) is rather surprising since formation of the activated compound (compound I) is very fast in the case of peroxidases. A tentative explanation may be derived from a kinetic analysis of the reactions of cytochrome c with HzOz. Since integrated kinetic equations for a consecutive series of reactions including reversible steps fail to give the concentration values as a function of time explicitly (20), activation and degradation of cytochrome c may be approached as two consecutive first-order reactions. As a rough approximation one can assume a reaction sequence represented by
where A, B, and C stand for ferricytochrome c, its activated form, and its degradation product, respectively. Defining now the pseudo-first-order constants k’, = kl [HzO,] and k; = kp [H202], the concentration of these species as a function of time are given by (21)
B=
$!j!$ 2
(&t
_
e-k;t)
1
and C=Ao-A-B.
[31
ET AL.
The maximum at the time t
value of B corresponds with dB/dt
1 = ___ (In k& - In kl,). nmx k’, - k;
= 0,
t41
Equation
[4] allows one to visualize the effect of varying It can be demonstrated that t,,, is inversely related to the absolute value of the difference k; - k’, . For example, multiplying both k; and k’, by an arbitrary value n gives k’, and k; on the value of t,,,.
t max=
1 n(kk
- k;)
(Ink;
- In k;).
]51
It must be emphasized that t,,, is not only related to the velocity of formation of B but also to the velocity of consumption of this species. The different behavior of cytochrome c and peroxidases may be due not only to a different velocity of compound I formation but also to a different k2 yielding, in the case of cytochrome c, a smaller difference in k’, - k;. Obviously, the actual reaction scheme may be more complex, involving reversible steps, isomerization, etc. More experiments are needed to allow a more accurate quantitative comparison. Elimination of the Soret absorbance at 408 nm indicates oxidation of the porphyrin ring, leading to opening of an cu-methene bridge (22). Nevertheless, free iron coming from heme degradation (23) did not play a role in catalysis of oxidative processes as concluded from the lack of effect of DTPA. Thus, participation of the protein moiety was essential for substrate oxidation. The lack of spectral evidence for the catalytically active form of cytochrome c is possibly due to its reactivity and short half-life, resulting in a very low steady-state concentration. While peroxidases are inactivated by HZ02, with cytochrome c the ratio between the rates of cytochrome c degradation and formation of the reactive oxidant species could be much higher. If the differences in peroxidatic activity observed between cytochrome c and horseradish peroxidase were related to the respective concentrations of active compounds, that of cytochrome c would be undetectable by spectrophotometry. Luminol chemiluminescence induced by hemeproteins and H20, has been previously recognized (24). Our results with cytochrome c suggest an alternative mechanism of chemiexcitation by hemeproteins. In the case of HRP, it was proposed that luminol is oxidized in a monovalent step to the luminol radical. This in turn can reduce molecular oxygen to 0,) with this species leading to the formation of an unstable luminol endoperoxide which decomposes with the emission of light (25). In our experiments SOD had no effect, excluding a role for 0; in cytochrome c-catalyzed, H20z-dependent luminol chemiexcitation. Moreover, the luminol chemilumines-
CYTOCHROME
c AND
cence observed in the absence of oxygen and the lack of 0; sensitive lucigenin photoemission (24) further supports that 0, is not an intermediate. We postulate that cytochrome c performs a two-electron oxidation of luminol to luminol diazoquinone, which in turn is dioxygenated directly by Hz02 to the excited species, luminol endoperoxide (26). Uric acid and tryptophan inhibition of cytochrome c/H,O,-induced luminol photoemission is a complex process. Both molecules have direct inhibitory effects on cytochrome c peroxidatic activity, but can also undergo reactions with luminol intermediates and interfere with chemiexcitation. For example, urate might prevent luminol chemiexcitation by reduction of luminol radical intermediates (27). Tryptophan only partially (15%) inhibited cytochrome c peroxidatic activity, but quenched almost completely light emission induced by the same system (Table II), in agreement with a primary effect on the chemiexcitation process rather than on catalytic activity. Finally, interference of urate and tryptophan on luminol radiative deactivation cannot be excluded. Hydrogen peroxide is a normal byproduct of mitochondrial and cellular metabolism (28) and is toxic to cells at elevated concentrations (29). The minute amount of iron bound to low molecular weight compounds (i.e., nucleotides, carboxylic acids) is usually considered to mediate HZOp-cytotoxic effects through the Fenton reaction (30). Interestingly, it has also been reported that myoglobin (31, 32) and hemoglobin (32,33) have pro-oxidant effects in the presence of H202 which may be of physiological relevance. In addition, we herein show that cytochrome c can participate in H,O,-mediated oxidation reactions. Thus, in future investigations it will be important to assess the quantitative contribution of nonprotein versus protein-bound iron to cell damage from reactive oxygen species.
HYDROGEN
117
PEROXIDE
5. Vandewalle, P. L., and Petersen, N. 0. (1987) FEBS Lett. 210, 195-198. 6. Turrens, J. F., and McCord, J. M. (1988) FEES Lett. 227, 43-46. I. Radi, R., Rubbo, H., and Prodanov, E. (1989) Biochim. Biophys. Acta
994,
89-93.
8. Green, M., and Hill, H. A. 0. (1984) in Methods in Enzymology (Packer, L., Ed.), Vol. 105, Academic Press, New York, 3-32. Photobiol. 18, 9. Hodgson, E. K., and Fridovich, I. (1973) Photochem. 451-455. Pho10. Merenyi, G., Lind, J., and Eriksen, T. E. (1985) Photochem.
tobiol. 41, 203-208. 11. Halliwell, B., and Gutteridge, J. M. C. (1981) FEBS Lett. 128,347352. 12. Oyamburo, G., Prego, C., Prodanov, E., and Soto, H. (1970) Biochim. Biophys. Acta 205, 190-195. 13. Gurney, R. W. (1953) Ionic Processes in Solution, pp. 80-112. McGraw-Hill, New York. 14. Dickerson, R., and Timkovich, R. (1975) in The Enzymes (Boyer, P. D., Ed) Vol. XI (A), pp. 397-547, Academic Press, San Diego. 15. Walsh, C. (1979) Enzymatic Reaction Mechanisms, pp. 4644500. Freeman, San Francisco. 16. Morishima, I., and Ogawa, S. (1979) J. Biol. C&m. 254,2814-2820. 17. Dunford, H. B. (1987) Free Rad. Biol. Med. 3, 405-421. C. (1989) Free Kadic. Biol. Med. 6, 18. Sutton, H., and Winterbourn, 53360. 19. Howell, R. R., and Wyngaarden, J. B. (1960) J. Biol. Chem. 235, 3544-3550. A. (1978) Principles of Enzyme Kinetics, Butter20. Cornish-Bowden, worths, London/Boston. 21. Frost, A. A., and Pearson, R. G. (1961) Kinetics and Mechanism. 2nd ed., pp. 166-169, Wiley New York/London. 22. O’Brien, P. J. (1966) Biochem. J. 101, 12P. 4, 23. Puppo, A., and Halliwell, B. (1988) Free Radic. Res. Common. 415-422. 24. Allen, R. C. (1982) Chemical and Biological Generation of Excited States, pp. 309-344 Academic Press, New York. 25. Misra, H. P., and Squatrito,
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ACKNOWLEDGMENTS We thank Dr. Bruce A. Freeman for his helpful comments during the preparation of this manuscript and Mrs. Susana Tolosa for her technical assistance. This work was supported by the Programa de Desarrollo de Ciencias Basicas (PEDECIBA), Uruguay.
27. Radi, R., Rubbo, H., Thomson, Radic. Bioi. Med. 8, 121-126.
L., and Prodanov,
E. (1990) Free
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OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 288, No. 1, July, pp. 112-117, 1991
Cytochrome c-Catalyzed Oxidation of Organic Molecules by Hydrogen Peroxide Rafael Radi,’ Leonor Thomson,
Homer0 Rubbo, and Eugenio Prodanov’
Department of Biochemistry, Faculty of Medicine, University Au. Gral. Flares 2125, Montevideo, Uruguay CP 11800
Received December 14, 1990, and in revised form February
of the Republic,
26, 1991
Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (HzO,), including 2-2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the HzOz-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with HzOz increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (0;) and hydroxyl radical (‘OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H202 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H,Oz-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of HzOz with an oxidized luminol molecule, either luminol radical or luminol diazoquinone. 0 issi Academic PESS, IN.
ported that cytochrome c participates in the hydroxylation of 4-nitrophenol (2) and the oxidation of 2-keto-4-thiomethyl butyric acid (3) in the presence of HzOz. On the other hand, cytochrome c is degraded by H202 and hydroperoxides, with irreversible spectral changes and production of spontaneous chemiluminescence (4). During the interaction of cytochrome c with H202, hydroperoxides, and reducing substrates, the mechanism of iron involvement and the participating active species have not been well defined. Cytochrome c2+ can be reoxidized to cytochrome c3+ by H202, giving rise possibly to ‘OH (5, 6). When cytochrome c3+ reacts with HzOz formation of a bound hydroxyl radical (Fe3+--‘OH) by a site-specific reaction at the heme was proposed (4). We have previously shown that ferricytochrome c increases luminol chemiluminescence produced by the xanthine oxidase/hypoxanthine/OZ system (7). Because cytochrome c3+ scavenges 0, (8) and luminol chemiluminescence is usually dependent on 0; (7, 9, lo), the light emission increase was unexpected and suggested a more complex role of cytochrome c. In this work we studied the reactions of cytochrome c with Hz02 and the secondary oxidation of organic molecules. Herein we report a kinetic analysis of this catalytic action of cytochrome c and concurrently communicate an alternative mechanism of luminol chemiexcitation by hemeproteins. These data suggest that cytochrome c might play a role in mediating or enhancing cytotoxic effects of HzOz. MATERIALS
AND
METHODS
While the peroxidatic activity of several hemoproteins and hematin is well understood (l), the mechanism and kinetics of cytochrome c-catalyzed oxidation of substrates by HzOz has not been explored in detail. It has been re-
Cytochrome c (types III and VI), luminol (5amino-2,3-dihydro-1,4phtalazinedione), lucigenin (his-N-methylacridinium nitrate), 2-2’-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS): 4-aminoantipyrine (4.AP), diethylene triaminopentaacetic acid (DTPA), 2thiobarbituric
i Current address: Departments of Anesthesiology and Biochemistry, The University of Alabama at Birmingham, 619 19th St. South, 941 THT, Birmingham, Alabama 35233. ’ To whom correspondence should be addressed.
3 Abbreviations used: ABTS, 2-2’.azino-bis (3-ethylbenzthiazoline-6sulfonic acid); 4-AP, 4-amino-antipyrine; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid; HRP, horseradish peroxidase.
112
0003.9861/91$3.00 All
Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
CYTOCHROME
c AND
acid and mannitol were obtained from Sigma. Hydrogen peroxide was obtained from Baker; uric acid and sodium dithionite were from Fisher. All other reagents used were of analytical grade. Cytochrome c was reduced by sodium dithionite and purified by chromatography on Sephadex G-25 using 10 mM potassium phosphate, pH 7.0, as the elution buffer. In some experiments cytochrome c3’ was also purified on Sephadex G-25 in order to remove potential hemopeptide residues with microperoxidase activity (6). No differences in the reactions catalyzed by cytochrome c were observed when compared to untreated cytochrome c3+. Spectrophotometric measurements were performed in a Shimadzu uv 160 spectrophotometer. Oxidation rates of 4-AP (6~ = 6.4 X 10” Mu ‘. cm-‘, Ref. (2)) and ABTS (tM = 3.6 X lo4 M-’ . cm- ‘, Ref. (10)) were followed at 505 and 420 nm, respectively. Hydroxyl radical formation was studied by deoxyribose degradation to 2.thiobarbituric acid reactive material (11). Chemiluminescent reactions were carried out under continuous oxygen bubbling as described before (7, 12). Nitrogen replaced oxygen in some cases for anaerobic studies. A 0.1 M pyrophosphate buffer, pH 8.3, was used for chemiluminescent reactions. For other assays, buffer systems were chosen which were effective at a particular pH range; to rule out remote buffer effects on catalytic activity, reaction rates were determined at each pH with two different buffer systems. None of the buffers reported in this manuscript significantly inhibited cyt,ochrome c/H,O,-induced ABTS and 4-AP oxidation. Each set of experiments reported herein was repeated a minimum of three times with similar results. Data represent the mean of two different analyses from a representative experiment.
RESULTS
Oxidation of ABTS and 4-AP by H,O, in the Presence of Cytochrome c ABTS and 4-AP were oxidized by H202 in the presence of cytochrome eni. Reactions obeyed Michaelis-Menten kinetics for H202, having K, values of 65 mM at pH 6.0 to 7.5 at saturating concentrations of the reductants, as determined by a Lineweaver-Burk plot (Fig. 1). This K, represents a binding energy of approximately 4.1 kcal/ mol for the interaction cytochrome c-HzOz, calculated as unitary free energy (13). Maximal dependence of velocity on ABTS concentration was found in a very narrow range correfrom 1 to 6 yM and the substrate concentration sponding to half-maximal reaction velocity was within the micromolar concentration range. The reaction velocity remained nearly constant when the ABTS concentration was higher than 10 PM. Second-order constants were 3 X 10’ [H202]. mini’ for ABTS and 4.1 X lo5 [ABTS] . min * for H,O,. Horseradish peroxidase (HRP) oxidized ABTS and 4-AP at lo4 to lo5 faster rates than equimolar cytochrome c”+, at pH 6.0. Oxidation rates were directly proportional to cytochrome c3+ concentration from 0.75 to 30 WM. When cytochrome es+ was used there was a lag phase before the oxidation of ABTS or 4-AP started. This lag phase corresponded to the time required to fully oxidize cytochrome c2+to the ferric state in the presence of HZ02, monitored spectroscopically as the disappearance of the 550-nm band (characteristic of cytochrome c2+, tM = 2,l X 10’ Mm’ * cm-‘; Ref. (8)). The second-order rate constant
HYDROGEN
113
PEROXIDE
c2+ reoxidation by HzOz was 125 cytochrome emin’, consistent with other observations (6). Cyanide inhibited the oxidation of ABTS and 4-AP by cytochrome c3+ and HaOz. Oxidation rates of ABTS in the presence of 6 PM cytochrome c3+ plus 0.73 or 4.4 mM H202 at pH 5.6 were inhibited by 60 and 45%, respectively, using 0.17 mM cyanide. The pH profile of the catalytic activity of cytochrome c is represented in Fig. 2. This activity was higher in the acidic pH range, with a maximum at pH 3.6-3.8. There was no activity toward ABTS and 4-AP at pH > 9.5. of
M-r
Catalytic Activity of Cytochrome c and Its Relation with Spectral Changes Brief preincubation of cytochrome c3+ with H,Oz increased catalytic activity; longer pre-incubation led to a progressive decrease (Fig. 3). At the same time, the spectra of cytochrome c showed a continuous exponential decrease of the Soret band (408-410 nm; not shown). Degradation of cytochrome c by H202 showed a second-order rate constant (h’) of 14.2 [H202] emin’ at 18”C, pH 6.0 (Table I). Diethylenetriaminepentaacetic acid, SOD, and mannitol added to the preincubation medium did not alter catalytic activity or the spectral changes. Tryptophan (5 mM) inhibited cytochrome c degradation and oxidation rates by approximately 15%, with uric acid having an even more potent inhibition. Uric acid influence on catalytic activity and cytochrome c degradation depended heavily on uric acid/H20Z molar ratios (Fig. 3, Table I). When uric acid:HpOl molar ratios were low (l:lO), no effects of uric acid were observed. With ratios around l:l, cytochrome c activation was delayed (Fig. 3) as well as inactivation (Fig. 3) and degradation (Table I). Uric acid in
-0.02
0.10
0.04 [H,OJ-’
0.16
hM)-’
FIG. 1. Michaelis constant (K,) determination for HzO,. Different concentrations of H,O, (6.7 to 80 mM) were incubated in the presence of 0.6 pM cytochrome c3+ and 20 mM ABTS in 0.067 M potassium phosphate, pH 7.0, at 18°C. Initial reaction rates were obtained by spectrophotometric measurement of ABTS oxidation.
1 14
RADI
ET AL. TABLE
0.05
Influence
of Uric
0.04
?
Acid and H,Os on Cytochrome Degradation Rates
[Uric acidJ/[H,Os]
E 0.03
t E
I
k’([H,O,]
0 0.1 0.4 1.0 5.0
>" 0.02
0.01
3
5
7
9
11
PH FIG. 2. Influence of pH on catalytic activity. Assay conditions were: 6 pM cytochrome c3+, 0.6 mM H202 and 1.3 mM ABTS at different pH, 18’C. The buffer systems used were 0.05 M phtalate-HCl, pH 2.0 to 5.0; 0.05 M citrate-phosphate, pH 5.4 to 7.0; 0.05 M pyrophosphate, pH 8.0 and 8.5; 0.05 M borate, pH 9.0 to 10.0.
c3+
.min-i)
14.2 14.2 14.2 11.1 4.7
Note. Cytochrome c 3t (67 FM) was incubated with 1.9 mM H202 and different uric acid concentrations in 0.067 M potassium phosphate, pH 6.0, at 18°C. At different time points aliquots were taken and diluted to a final concentration of 6.7 @M cytochrome c for spectroscopic measurements. Spectral changes were followed by the disappearance of Soret band absorbance at 408 nm. Data represents X (n = 2) from a representative experiment which had a maximum variation of 0.02 [H,O,] mini between replicates.
4). There was a lag phase and a lower intensity peak of Chemilucytochrome c2+-induced chemiluminescence. excess over H202 (5:l) completely suppressed catalytic minescent yields and lag phases were dependent on HzOz activity and substantially inhibited cytochrome c degraconcentration (Fig. 5). Lag phases of photoemission were dation (Table I). shortened and even disappeared if cytochrome c2+ was No 2-thiobarbituric acid reactive material was detected after deoxyribose was incubated with cytochrome c3+ preincubated with HzOz. The dependence of chemiluminescence on luminol concentration is shown in Fig. 6. and H202. Initial velocities of chemiluminescence increase (ucL) were measured as the initial slope of light intensity versus time. Chemiluminescence Produced by a Cytochrome Data were fitted to a Lineweaver-Burk plot with an apc-H202-Luminol System parent K,,, of 84 PM. Both ferro and ferric forms of cytochrome c were caHorseradish peroxidase concentrations equimolar with pable of producing luminol chemiluminescence in the cytochrome c induced a very strong luminol-dependent presence of H20z, but with different time courses (Fig. light emission, in the order of lo3 bigger than that of cy-
1200
900
.L 0
10 preincubatlon
20 time
30
7--
0
a
10
20
30
(mid
FIG. 3. Influence of preincubation with H,O, on catalytic activity. Cytochrome c3+ (83 pM) was pr eincubated with 0.6 mM H202 in the absence (0) or presence (0) of 0.4 mM uric acid in 0.067 M potassium phosphate at 18°C. At different times aliquots were added to 27 mM 4AP. Final concentrations of cytochrome c and H,Oz after dilution were 9 pM and 0.065 mM, respectively. Initial reaction rates were obtained by following 4-AP oxidation spectrophotometrically.
time
(min)
FIG. 4. Time course of cyt c/HsO*-induced luminol chemiluminescence. Reagent concentrations were: cytochrome c3+ (A) or cytochrome c*+ (0) 25 pM and HRP (0) 25 nM; lumino10.67 mM and HzOs, 0.6 mM in 0.1 M pyrophosphate pH at 18°C. Reactions were initiated by addition of the cytochrome. Light intensity was measured in photometer scale units.
CYTOCHROME
c AND
HYDROGEN
115
PEROXIDE
4
600
3 400
L
,x 2 a, .-E E .P 200
0 0 I
5
IO
15
20
OL -20 25
hnird-’
time (min)
FIG. 5. Time course of luminol chemiluminescence as a function of H,Os concentration. Concentrations of the reagents were: cytochrome I?, 30 PM; luminol, 0.67 mM, and H202, 0.15 mM (e), 0.30 mM (W), 0.45 mM (O), 0.60 mM (A.). Reactions were conducted in 0.10 M pyrophosphate, pH 8.3, at 18°C. Light intensity was measured in photometer scale units.
tochrome c, which decayed very rapidly (~15 s) due to HzOz depletion. When HRP was diluted lo3 times, chemiluminescence yield was similar to that induced by cytochrome c, without a lag phase (Fig. 4). Luminol chemiluminescence induced by cytochrome c/H202 was inhibited instantaneously and completely by 1 mM uric acid, almost completely by 1 mM tryptophan, with SOD and mannitol having no effect (Table II). No luminol chemiluminescence was observed when uric acid was present at the beginning of the reaction. The chemiluminescent probe, lucigenin, gave a very weak light emission with a maximal intensity of about 0.2% of that of luminol. No dependence upon oxygen concentration was found for catalytic activity toward reducing substrates, including luminol (not shown).
knM)-’
FIG. 6. Influence of luminol concentration on chemiluminescence. The reaction mixtures contained 30 PM cytochrome c3+, 0.6 mM H202 and different luminol concentrations (0.02 to 0.66 mM) in 0.1 M pyrophosphate, pH 8.3, at 18’C. Rates of luminol oxidation were determined as the initial slope of light intensity (photometer scale units) versus time (seconds).
high K,,, for H202 indicate that the donor molecules bind to cytochrome c at sites other than the ferric heme iron. Peroxidases bind their electron donor substrates in hydrophobic clefts in close vicinity to the heme moiety (15, 16). Binding to a cytochrome c hydrophobic cleft (involving multiple noncovalent hydrophobic interactions) is in agreement with high affinities found for reducing substrates. An interesting problem concerns the mechanism of substrate oxidation. Ferrous iron was not involved, because with ferrocytochrome c a lag phase preceded the beginning of the peroxidatic activity, corresponding to the oxidation of the ferrous to the ferric form of cytochrome c. With ferricytochrome c, no lag time was observed, although there was often an acceleration before
DISCUSSION
Our results support a catalytic mechanism for the oxidation of organic molecules by cytochrome c in the presence of Hz02. Data are consistent with a Michaelis-Menten kinetics for HzOz having a K, of approximately 65 mM (Fig. 1). This K, reflects a very weak affinity for HzOz, consistent with a nonionic and noncovalent bond of approximately 4.1 kcal/mol between HzOz and cytochrome c. Inhibition of ABTS and 4-AP oxidation by cyanide supports the idea that catalysis by cytochrome c involves the sixth ligand position of the iron in binding HA (4, 14). There is a low specificity of cytochrome c toward electron donors as demonstrated herein and elsewhere (2, 3) but the kinetic data supports a saturation behavior. The reductants appeared to bind to cytochrome c as shown by the K, value for luminol and the Ko.5for ABTS. The low Km found for the electron donors in comparison to the
TABLE
II
Effect of Free Radical Scavengers on Cytochrome c3+Plus H,Oz-Induced Luminol Chemiluminescence Reagent None Urate Tryptophan Mannitol SOD
Light intensity 120 0 15 120 120
% Inhibition
100 87 0 0
Note. Cytochrome c3+ (10 PM) was incubated with 0.84 mM H,O, and 0.067 mM luminol in 0.1 M pyrophosphate, pH 8.3,18”C. A steady-state chemiluminescence of 120 photometer scale units was obtained by 20 min. At t = 23 min, 1 mM mannitol, 1 mM tryptophan, 1 mM urate, or 1 PM SOD was added. Data reports inhibition of photoemission immediately after addition of scavengers; in all cases percentage inhibition remained constant during a 15-min observation period.
116
RADI
attaining a steady-state. The fact that catalytic activity increased after a short preincubation of ferricytochrome c with hydrogen peroxide (Fig. 3) strongly suggests that ferricytochrome c was not the actual active form but a precursor. Our experiments give no direct evidence for the oxidation state of iron, but the lack of effect of SOD makes it very unlikely to be a catalyzed Haber-Weiss type mechanism. Lag times obtained with ferrocytochrome c, insensitivity to mannitol, and absence of deoxyribose degradation exclude a Fenton-like reaction producing ‘OH radical. Therefore, the catalytically active form of cytochrome c must bear iron in a more oxidized state, as in compound I of peroxidases where a ferry1 (Fe4+=0) ion is bound to a porphyrin cation radical. Ferryl ion has similar reactivity and is kinetically indistinguishable from ‘OH (17, 18). Formation of the ferry1 species is in good agreement with the inhibitory effects of uric acid in either substrate oxidation or cytochrome c degradation since uric acid is an excellent scavenger of oxo-heme oxidants (19), competing for the oxidant with the substrates or the sensitive groups of the protein. ln the case of cytochrome c, however, this high oxidation state protein species must be very unstable and would be easily degraded by reaction with HzOz or by internal rearrangement of the oxidized molecule. The fact that maximum activity is reached after a definite time interval (Fig. 3) is rather surprising since formation of the activated compound (compound I) is very fast in the case of peroxidases. A tentative explanation may be derived from a kinetic analysis of the reactions of cytochrome c with HzOz. Since integrated kinetic equations for a consecutive series of reactions including reversible steps fail to give the concentration values as a function of time explicitly (20), activation and degradation of cytochrome c may be approached as two consecutive first-order reactions. As a rough approximation one can assume a reaction sequence represented by
where A, B, and C stand for ferricytochrome c, its activated form, and its degradation product, respectively. Defining now the pseudo-first-order constants k’, = kl [HzO,] and k; = kp [H202], the concentration of these species as a function of time are given by (21)
B=
$!j!$ 2
(&t
_
e-k;t)
1
and C=Ao-A-B.
[31
ET AL.
The maximum at the time t
value of B corresponds with dB/dt
1 = ___ (In k& - In kl,). nmx k’, - k;
= 0,
t41
Equation
[4] allows one to visualize the effect of varying It can be demonstrated that t,,, is inversely related to the absolute value of the difference k; - k’, . For example, multiplying both k; and k’, by an arbitrary value n gives k’, and k; on the value of t,,,.
t max=
1 n(kk
- k;)
(Ink;
- In k;).
]51
It must be emphasized that t,,, is not only related to the velocity of formation of B but also to the velocity of consumption of this species. The different behavior of cytochrome c and peroxidases may be due not only to a different velocity of compound I formation but also to a different k2 yielding, in the case of cytochrome c, a smaller difference in k’, - k;. Obviously, the actual reaction scheme may be more complex, involving reversible steps, isomerization, etc. More experiments are needed to allow a more accurate quantitative comparison. Elimination of the Soret absorbance at 408 nm indicates oxidation of the porphyrin ring, leading to opening of an cu-methene bridge (22). Nevertheless, free iron coming from heme degradation (23) did not play a role in catalysis of oxidative processes as concluded from the lack of effect of DTPA. Thus, participation of the protein moiety was essential for substrate oxidation. The lack of spectral evidence for the catalytically active form of cytochrome c is possibly due to its reactivity and short half-life, resulting in a very low steady-state concentration. While peroxidases are inactivated by HZ02, with cytochrome c the ratio between the rates of cytochrome c degradation and formation of the reactive oxidant species could be much higher. If the differences in peroxidatic activity observed between cytochrome c and horseradish peroxidase were related to the respective concentrations of active compounds, that of cytochrome c would be undetectable by spectrophotometry. Luminol chemiluminescence induced by hemeproteins and H20, has been previously recognized (24). Our results with cytochrome c suggest an alternative mechanism of chemiexcitation by hemeproteins. In the case of HRP, it was proposed that luminol is oxidized in a monovalent step to the luminol radical. This in turn can reduce molecular oxygen to 0,) with this species leading to the formation of an unstable luminol endoperoxide which decomposes with the emission of light (25). In our experiments SOD had no effect, excluding a role for 0; in cytochrome c-catalyzed, H20z-dependent luminol chemiexcitation. Moreover, the luminol chemilumines-
CYTOCHROME
c AND
cence observed in the absence of oxygen and the lack of 0; sensitive lucigenin photoemission (24) further supports that 0, is not an intermediate. We postulate that cytochrome c performs a two-electron oxidation of luminol to luminol diazoquinone, which in turn is dioxygenated directly by Hz02 to the excited species, luminol endoperoxide (26). Uric acid and tryptophan inhibition of cytochrome c/H,O,-induced luminol photoemission is a complex process. Both molecules have direct inhibitory effects on cytochrome c peroxidatic activity, but can also undergo reactions with luminol intermediates and interfere with chemiexcitation. For example, urate might prevent luminol chemiexcitation by reduction of luminol radical intermediates (27). Tryptophan only partially (15%) inhibited cytochrome c peroxidatic activity, but quenched almost completely light emission induced by the same system (Table II), in agreement with a primary effect on the chemiexcitation process rather than on catalytic activity. Finally, interference of urate and tryptophan on luminol radiative deactivation cannot be excluded. Hydrogen peroxide is a normal byproduct of mitochondrial and cellular metabolism (28) and is toxic to cells at elevated concentrations (29). The minute amount of iron bound to low molecular weight compounds (i.e., nucleotides, carboxylic acids) is usually considered to mediate HZOp-cytotoxic effects through the Fenton reaction (30). Interestingly, it has also been reported that myoglobin (31, 32) and hemoglobin (32,33) have pro-oxidant effects in the presence of H202 which may be of physiological relevance. In addition, we herein show that cytochrome c can participate in H,O,-mediated oxidation reactions. Thus, in future investigations it will be important to assess the quantitative contribution of nonprotein versus protein-bound iron to cell damage from reactive oxygen species.
HYDROGEN
117
PEROXIDE
5. Vandewalle, P. L., and Petersen, N. 0. (1987) FEBS Lett. 210, 195-198. 6. Turrens, J. F., and McCord, J. M. (1988) FEES Lett. 227, 43-46. I. Radi, R., Rubbo, H., and Prodanov, E. (1989) Biochim. Biophys. Acta
994,
89-93.
8. Green, M., and Hill, H. A. 0. (1984) in Methods in Enzymology (Packer, L., Ed.), Vol. 105, Academic Press, New York, 3-32. Photobiol. 18, 9. Hodgson, E. K., and Fridovich, I. (1973) Photochem. 451-455. Pho10. Merenyi, G., Lind, J., and Eriksen, T. E. (1985) Photochem.
tobiol. 41, 203-208. 11. Halliwell, B., and Gutteridge, J. M. C. (1981) FEBS Lett. 128,347352. 12. Oyamburo, G., Prego, C., Prodanov, E., and Soto, H. (1970) Biochim. Biophys. Acta 205, 190-195. 13. Gurney, R. W. (1953) Ionic Processes in Solution, pp. 80-112. McGraw-Hill, New York. 14. Dickerson, R., and Timkovich, R. (1975) in The Enzymes (Boyer, P. D., Ed) Vol. XI (A), pp. 397-547, Academic Press, San Diego. 15. Walsh, C. (1979) Enzymatic Reaction Mechanisms, pp. 4644500. Freeman, San Francisco. 16. Morishima, I., and Ogawa, S. (1979) J. Biol. C&m. 254,2814-2820. 17. Dunford, H. B. (1987) Free Rad. Biol. Med. 3, 405-421. C. (1989) Free Kadic. Biol. Med. 6, 18. Sutton, H., and Winterbourn, 53360. 19. Howell, R. R., and Wyngaarden, J. B. (1960) J. Biol. Chem. 235, 3544-3550. A. (1978) Principles of Enzyme Kinetics, Butter20. Cornish-Bowden, worths, London/Boston. 21. Frost, A. A., and Pearson, R. G. (1961) Kinetics and Mechanism. 2nd ed., pp. 166-169, Wiley New York/London. 22. O’Brien, P. J. (1966) Biochem. J. 101, 12P. 4, 23. Puppo, A., and Halliwell, B. (1988) Free Radic. Res. Common. 415-422. 24. Allen, R. C. (1982) Chemical and Biological Generation of Excited States, pp. 309-344 Academic Press, New York. 25. Misra, H. P., and Squatrito,
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ACKNOWLEDGMENTS We thank Dr. Bruce A. Freeman for his helpful comments during the preparation of this manuscript and Mrs. Susana Tolosa for her technical assistance. This work was supported by the Programa de Desarrollo de Ciencias Basicas (PEDECIBA), Uruguay.
27. Radi, R., Rubbo, H., Thomson, Radic. Bioi. Med. 8, 121-126.
L., and Prodanov,
E. (1990) Free
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