ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 288, No. 1, July, pp. 118-125, 1991

Cytochrome c-Catalyzed by Hydrogen Peroxide Rafael Radi,‘,* Julio F. Turrens,?

Membrane

Lipid Peroxidation

and Bruce A. Freeman2’*

*Departments of Anesthesiology, Biochemistry and Pediatrics, The University of Alabama at Birmingham, Birmingham, 35233; and the tDepartment of Biomedical Sciences, University of South Alabama, Mobile, Alabama 36688

Received December 14, 1990, and in revised form February

Alabama

26, 1991

Cytochrome c3+-catalyzed peroxidation of phosphatidylcholine liposomes by hydrogen peroxide (H,O,) was indicated by the production of thiobarbituric acid reactive substances, oxygen consumption, and emission of spontaneous chemiluminescence. The iron chelator diethylenetriaminepentaacetic acid (DTPA) only partially inhibited peroxidation when HzOz concentrations were 200 PM or greater. In contrast, iron compounds such as ferric chloride, potassium ferricyanide, and hemin induced H,Oz-dependent lipid peroxidation which was totally inhibitable by DTPA. Cyanide and urate, which react at or near the cytochrome-heme, completely prevented lipid peroxidation, while hydroxyl radical scavengers and superoxide dismutase had very little or no inhibitory effect. Changes in liposome surface charge did not influence cytochrome c3’ plus HaOZ-dependent peroxidation, but a net negative charge was critical in favoring cytochrome c3’-dependent, H,Oz-independent lipid auto-oxidative processes. These results show that reaction of cytochrome c with H202 promotes membrane oxidation by more than one chemical mechanism, including formation of high oxidation states of iron at the cytochrome-heme and also by heme iron release at higher Hz02 concentrations. Cytochrome c3+ could react with mitochondrial H202 to yield “site-specific” mitochondrial membrane lipid peroxidation during tissue oxidant C 1991 Academic Press, Inc. stress.

Hydrogen peroxide (H20& is an intracellular oxygen metabolite formed by superoxide anion (0;) dismutation (1) or directly via two-electron reduction of oxygen. Partially reduced intermediates in the reduction of 0, to HZ0 I Permanent address: Department of Biochemistry, School of Medicine, University of the Republic, Montevideo, Uruguay, CP 11800. ‘To whom correspondence should be addressed at Department of Anesthesiology, The University of Alabama at Birmingham, 619 19th St. South, Birmingham, Alabama 35233.

may be produced by a variety of biological processes including direct transfer of electrons to O2 by enzymes (2), from intermediates in electron transport systems (i.e., ubisemiquinone or Fe-S centers of NADH dehydrogenase; Ref. (3)), or by auto-oxidation of molecules such as ascorbate or thiols (4). Hydrogen peroxide is hazardous to cells because it can exert cytotoxic effects by oxidizing critical biomolecules (5). For this reason, the intracellular steady-state concentration of H202 is kept low (lo-’ to 10m7M) by ubiquitous antioxidant systems based on two enzymes, catalase and glutathione peroxidase (6). However, cell HzOz concentrations can increase severalfold secondary to pathological events including hyperoxia (7, 8), ischemia-reperfusion (9), and inflammation (10). Membrane lipid peroxidation has been recognized as a major deleterious effect of increased intracellular H,02 production (11). Nevertheless, HzO, has a very low reactivity toward unsaturated fatty acids, with HeOz-induced lipid peroxidation requiring metal catalysis in order to occur at biologically significant rates (12). Ferrous iron has been proposed as the main chemical species responsible for reducing HaOz to HZ0 and the reactive hydroxyl radical (‘OH) in a Fenton-like reaction (12). Hydroxyl radical reacts with biomolecules such as unsaturated fatty acids with a second-order rate constant of about 10’ -1 -1 ‘S , near diffusion controlled rates (13). Lipid perM oxidation is induced by ‘OH via hydrogen abstraction, at carbon atoms in the CYposition with respect to the olefin (12). Because hydroxyl radical is so reactive, it will react with molecules within a radius of 3 to 5 of its molecular diameter. Thus, in order to initiate lipid peroxidation, ‘OH must be generated vicinal to unsaturated membrane lipids (14). It has been proposed that metal catalysis of membrane lipid peroxidation occurs secondary to metal binding to membrane surfaces (15,16) or by partitioning into the bulk lipid phase (14). Iron in biological systems does not exist in free form, being complexed to protein or as chelates of low molecular weight anions (17,lS). Cellular reductants such as 0, can

118 All

0003.9861/91 83.00 Copyright D 1991 by Academic Press, Inc. rights of reproduction in any form reserved.

CYTOCHROME

c AND

cause iron mobilization from ferritin, the main iron-storage protein in cells (19). Low molecular weight iron chelates such as nucleotide-iron (i.e., ATP-Fe) or carboxylic acid-iron (i.e., citrate-Fe) complexes have been suggested as primary sources for iron-catalyzed reactions (20, 21). However, increasing evidence suggests that protein-associated iron participates in the activation of H202 to ‘OH or species of similar chemical reactivity, such as ferry1 (Fe4+= 0) or perferryl (Fe5’=O) iron (22-24). In support of this, cytochrome c plus Hz02 yields strong oxidant species, inducing spontaneous (26) and luminol-amplified chemiluminescence, indicating free radical production (24, 25). Cytochrome c3+ plus H202 will oxidize 2,2’-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS)3 (24), 4aminoantipyrine (24), 2-nitrophenol (27), and ketothiomethyl butyric acid (28), indicating the generation of ‘OH or ‘OH-like species. Cytochrome c is a mitochondrial intermembrane protein which binds to the inner membrane. Mitochondria are an important source of Hz02, which can either be scavenged intramitochondrially, react with mitochondrial target molecules, or diffuse to the cytosolic compartment (29). We show herein, using liposomes, that cytochrome C ‘+ and H202 react, producing strong oxidants that can attack polyunsaturated fatty acids and which might contribute to “site-specific” mitochondrial membrane lipid peroxidation. Additionally, oxidative modification of cytochrome c is seen to enhance its ability to mediate membrane oxidation. MATERIALS

AND

METHODS

Materials. Cytochrome c3+ (type III), 2-thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), diethylenetriaminepentaacetic acid (DTPA), urate, hemin, potassium ferricyanide, and mannitol were from Sigma. Ferric chloride and dimethylsulfoxide were obtained from Mallinckrodt, benzoate was from Merck, hydrogen peroxide was from Fluka, soybean phosphatidylcholine (soybean PC) was from Avanti Polar Lipids, 1,1,3,3-tetramethoxypropane was from Aldrich, and bovine CuZn superoxide dismutase (SOD) was a generous gift from Grunenthal, GmBH. Liposome preparation. Soybean phosphatidylcholine (PC) liposomes were prepared using 4.0 ml of a 25.4 mM (20 mg/ml) lipid stock solution in CHCl,. Solvent was removed in V~CUOat 45-50°C and 4.0 ml of 10 mM potassium phosphate, pH 7.4, was added. The suspension was placed in a 4°C water bath and sonicated 3 X 30 s at 65 W using a Branson sonifier. Liposomes were stored in the dark under an argon atmosphere and used within 24 h. Liposome membrane surface charge was changed by adding either anionic dicetylphosphate or cationic stearylamine in a 1O:l (mol/mol) ratio of phospholipid to amphiphile. Reaction systems. Hydrogen peroxide was prepared by dilution of was 10 M H,O, in deionized H,02. Hydrogen peroxide concentration assessed by measuring A,,,, nm (Ed = 43 Mm’ cm-‘). Liposome oxidation reactions were initiated by addition of H,O, after a 30-s incubation of

LIPID

119

PEROXIDATION

liposomes with cytochrome c3+ m 10 mM potassium phosphate, pH 7.4, at 37“C. Parallel control reactions in the absence of H,O, or cytochrome c were performed. For cytochrome c binding studies, liposomes were incubated with cytochrome c3+ for 5 min in 10 mM potassium phosphate, pH 7.4, at 37”C, then centrifuged 50 min at 15O,OOOg,4°C. Unbound cytochrome c was measured by Ass nm of supernatant after reduction with sodium dithionite (tM = 21,000 Mm’ cm-‘). Cytochrome c3’ was purified by chromatography on Sephadex G-25 to remove potential hemopeptide microperoxidase contamination (30). Biochemical analyses. Malondialdehyde (thiobarbituric acid reactive substances, TBARS) in liposome suspensions was determined by reaction with thiobarbituric acid using fluorescence spectroscopy (31, 32), thus avoiding interference due to cytochrome c absorbance in the same spectral region where the (TBA),-MDA chromophore absorbs. Fluorescent measurements were carried out using a 2-nm slit width, with X,, = 532 nm and X,, = 551 nm. Spectral parameters were determined from wavelength scans calibrated with known amounts of MDA obtained from acid hydrolysis of 1,1,3,3 tetramethoxypropane in 20% acetic acid, pH 3.5, using an SLM DMX-1000 fluorometer. Cytochrome c did not significantly quench fluorescence of the (TBA),-MDA adduct. To prevent further peroxidation of lipid during assay procedures, thiobarbituric acid reagent was made 0.025% with BHT. Low level chemiluminescence was measured at 30°C in a photon counter as described previously (33). Oxygen consumption was measured at 37°C using a Clark electrode, Model YSI 4004 (Yellow Spring Inst.) mounted in a l&ml water jacketed chamber.

RESULTS

Cytochrome c3+/H202-Induced Lipid Peroxidation No significant lipid peroxidation occurred when up to 1 mM H,O, alone was added to liposomes. Further addition of cytochrome c3+ resulted in lipid peroxidation (Fig. 1). Lipid peroxidation in liposome samples having no DTPA was dependent on H,O, concentrations up to 1 mM. Lipid peroxidation was strongly iron dependent, as evidenced by DTPA inhibition of TBARS formation (Fig. l), with DTPA-supplemented samples reaching maximal yields of

‘I

0.0

0.2

0.4

[H,OJ ’ Abbreviations used: DTPA, diethylenetriaminepentaacetic acid; TBA, thiobarbituric acid; BHT, butylated hydroxytoluene; PC, phosphatidylcholine; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive material; MDA, malondialdehyde; ABTS, 2-2’-azino-bis (3ethylbenzthiazoline-6.sulfonic acid).

0.6

0.0

1.0

(mM)

FIG. 1. Liposome lipid peroxidation as a function of H,O, concentration. Hydrogen peroxide was added to liposomes (2.6 pmol lipid phosphate/ml) plus cytochrome c3’ (10 KM) in the presence (0) or absence (0) of 20 pM DTPA in 10 mM potassium phosphate, pH 7.4. Reaction mixtures were incubated for 60 min at 37°C.

120

RADI,

0

10

[cytochrome

20

c]

TURRENS.

AND

FREEMAN

30

(,uM)

FIG. 2. Liposome lipid peroxidation as a function of cytochrome c concentration. Cytochrome c3+ was added to liposomes (2.6 lmol lipid phosphate/ml) plus 500 pM H202 in the presence of either 20 pM DTPA (0) or 100 FM DTPA (W) in 10 mM potassium phosphate, pH 7.4. Reaction mixtures were incubated for 60 min at 37°C. 10

0

20

30

40

50

60

time (min)

lipid peroxidation at 200 PM H,O, (Fig. 1). Lipid peroxidation yields were also affected by cytochrome c concentration (Fig. 2). There was generally a linear relationship between TBARS formation and cytochrome c3+ concentration. When DTPA was not in excess over cytochrome c3+, TBARS yield was higher than that predictable from the linear relationship between lower concentrations of cytochrome c3+ and TBARS formation (see arrow, Fig. 2). No cytochrome c-dependent, HzO,-independent autooxidation of neutrally charged phosphatidylcholine liposomes was detected. When ATP replaced DTPA as the iron chelator, peroxidation yields induced by 100 PM H,O, were significantly greater than in the absence of DTPA (2.44 k 0.09 versus 1.76 ? 0.06 PM, n = 3 f SD). Cytochrome c/HzOz-induced lipid peroxidation which was resistant to DTPA inhibition was examined further using different iron compounds. In the presence of 100 PM DTPA, neither hemin, potassium ferricyanide, nor ferric chloride (all 10 PM), initiated HzO,-dependent lipid oxidation, in contrast to cytochrome c. Hemin (but not potassium ferricyanide or ferric chloride) caused H,Ozindependent liposomal lipid auto-oxidation, producing about 5 pM TBARS (not shown). Kinetics of Lipid Oxidation There was a lag phase in lipid oxidation that depended on both H,Oz and cytochrome c3+ concentrations (Fig. 3). Increasing concentrations of either H,O, or cytochrome c3+ proportionally reduced the lag phase of TBARS formation. Once initiated, TBARS formation continuously increased during the l-h incubation period. The lag phase before onset of lipid oxidation was also verified polarographically. When 200 PM HzOz was added to liposomes (5.6 pmol lipid phosphate/ml) plus cyto-

FIG. 3. The effect of cytochrome c and H,OZ on the lag phase of liposome lipid peroxidation. Assay conditions were 10 pM cytochrome c3’ plus 40 WM (m) or 170 FM (+) H,O, (A) and 100 fiM H,OB plus 5 PM (A) or 10 pM (0) cytochrome ca’ ( B) All reactions had liposomes (2.6 pmol lipid phosphate/ml) in 10 mM potassium phosphate plus 100 PM DTPA, pH 7.4, at 37°C.

chrome c3+ (10 PM) in phosphate buffer, pH 7.4, with 20 yM DTPA, oxygen consumption began after approximately 5 min, reaching a maximum and stable rate of 1.3 nmol f min-’ . mll’ 12 min after the reaction started (Fig. 4). The lag phase of liposomal oxygen consumption was also dependent on both cytochrome c3+ and H202 concentration. No oxygen consumption above background

t-LO, 10 PM

1

Consumption O*

1

f 5 min b FIG. 4. Oxygen consumption induced by cytochrome c/H,O,-dependent lipid peroxidation. Hydrogen peroxide (200 pM) was added to liposomes (5.6 pmol lipid phosphate/ml) and 10 pM cytochrome c in 10 mM potassium phosphate plus 20 @M DTPA, pH 7.4, at 37°C.

CYTOCHROME TABLE

c AND

LIPID

121

PEROXIDATION

I

Cytochrome c3+Binding to Liposomes nmol cytochrome

3-

c bound s

Gmol liposome

’

phospholipid $2-

Liposome composition

lo

/.LM Cyt

C

20

PM

cyt

c 2

Phosphatidylcholine Phosphatidylcholine:stearylamine (lO:l, mol/mol) Phosphatidylcholinedicetylphosphate (lO:l, mol/mol) Phosphatidylcholine:dicetylphosphate (lO:l, mol/mol) + 150 NaCl

0.23

0.23

0.19

0.19

0.77

1.12

0.35

0.46

t

1 -

0

0.00

0.05

[H,O,] Soybean PC liposomes (2.6 pmol lipid phosphate/ml) were incubated with cytochrome ciit and its binding quantitated. NaCl was added to liposomes prior to cytochrome cat. Data represents n (n = 2) from a representative experiment that had a maximum variation of 0.03 nmol cyt c/nmol phospholipid between replicates. Note.

was observed in liposome suspensions in the absence of Hz02. Also, no significant oxygen consumption by cytochrome c3+ plus H202 in the absence of liposomes was detected. Effect of Membrane

Charge

In order to determine the effect of liposome-cytochrome c binding on H20,-dependent lipid oxidation, liposomes having different net surface charges and the effect of ionic strength were studied. Cytochrome c binding to liposomes was influenced by changes in liposome phospholipid composition (Table I). Negatively charged (PC-dicetylphosphate) liposomes bound the greatest amount of cytochrome czi+ per mole liposomal phospholipid (Table I). Elevation of the ionic strength by addition of 0.15 M NaCl to dicetylphosphate-containing liposomes diminished electrostatic interactions and displaced about 55-60% of bound cytochrome c. There was no direct relationship observed between the extent of cytochrome c binding to liposomes of differing phospholipid composition and their capacity to support HzO,-dependent lipid oxidation (Table I, Fig. 5). Only negatively charged liposomes supported cytochrome c-dependent, H,Op-independent auto-oxidation. The increase in MDA formation as a function of added HzOz was similar for all liposome preparations (Fig. 5). When liposomes were preincubated with isotonic NaCl before addition of cytochrome c3+, cytochrome c”+-dependent auto-oxidation of dicetylphosphate liposomes was inhibited more than 80% (Table II). In spite of this, isotonic NaCl did not prevent cytochrome c’+ plus H,02dependent peroxidation of negatively charged liposomes (Table II). Cytochrome

c Degradation

and Lipid

Peroxidation

The disappearance of Soret absorbance at 409 nm was used as an indicator of cytochrome c3+ heme degradation

0.15

0.10

0.20

(mM)

FIG. 5. Influence of liposomal-charge on lipid peroxidation yields. Hydrogen peroxide was added to liposomes (2.6 pmol lipid phosphate/ ml) plus cytochrome c” in 10 mM potassium phosphate plus 20 pM DTPA, pH 7.4. Liposomal composition was phosphatidylcholine (U), phosphatidylcholine:stearylamine (lO:l, mol/mol) (A) and phosphatidylcholinedicetylphosphate (lO:l, mol/mol) (0). Reaction mixtures were incubated for 60 min at 37°C.

(Table III). Cytochrome c3+ degradation by HzOz was indicated by DTPA-insensitive spectral changes which began immediately after H,Oz addition, either in the presence or absence of liposomes (Fig. 6). Cytochrome c degradation gave rise to TBARS which correlated well with Soret absorbance disappearance (Table III). TBARS yield from cytochrome c3+ reaction with 100-500 PM H202 increased proportionally with cytochrome c3’ concentration, using up to 30 PM cytochrome c3+ (data not shown). The spectroscopic characteristics of the fluorophore coming from H*O,-dependent cytochrome c3+ degradation were identical to the (TBA),-MDA adduct, with an iden-

TABLE

II

Effect of Isotonic NaCl on Cytochrome c-Catalyzed Lipid Peroxidation of Dicetylphosphate-Containing Liposomes TRARS Condition Control +cyt c +cyt c, H20L

(PM)

~ NaCl

+ NaCl

0.35 * 0.11 2.16 +- 0.16“ 4.20 4 0.09”

0.39 i- 0.22 0.72 k 0.13’ 3.43 f 0.28hJ

Note. Dicetyl phosphate-containing liposomes (2.6 rmol lipid phosphate/ml) were incubated for 1 h at 37°C with 10 pM cytochrome c3’ with or without 200 pM H,O, in 10 mM potassium phosphate, 20 pM DTPA, pH 7.4. Data represents X k SD, n = 3. e Significantly different from control without NaCl. b Significantly different from control with N&l. ‘Significantly different from matched treatment group having no NaCl, after ANOVA and analysis by Duncan’s multiple range test (P < 0.05).

122

RADI. TABLE

TURRENS.

AND

FREEMAN

III

time (min)

Cytochrome c3+ Degradation by H20, *.z;

WG&l (mM)

WARS1 (/IM)

0 0.1 0.5 1.0

0 0.04 0.14 0.21

A 409 0.90 0.75 0.28 0.17

Note. Cytochrome c3+ 10 pM was incubated at 37°C for 1 h in 10 mM potassium phosphate, 100 FM DTPA, pH 7.4. Cytochrome c degradation was measured by A 409and TBARS production. Data represent X (n = 2) from a representative experiment which had a maximum variation of 0.02 pM TBARS or 0.01 A,,, between replicates.

tical excitation and emission spectra (data not shown). The cytochrome c-derived TBARS were not due to lipid contamination of protein preparations and represented 2-1096 of the TBARS formed from liposome lipid peroxidation (Fig. 7). Potassium cyanide and urate substantially reduced cytochrome c3+ degradation and completely inhibited cytochrome c3+-dependent lipid peroxidation (Table IV). Dimethylsulfoxide, benzoate, and SOD exhibited little or no protection to cytochrome c3’ degradation and did not significantly inhibit liposomal oxidation. Cytochrome c3+ reaction with 500 PM H202 was accompanied by spontaneous chemiluminescence. Cytochrome c-catalyzed, HzO,-dependent lipid peroxidation also emitted low level light (Fig. 7). Under our experimental conditions direct reaction of Hz02 with cytochrome c3+ represented most of the photoemission, since chemiluminescence was minimally enhanced by liposome addition (Fig. 7). 1.00 ,

- lip0

+ lip0

FIG. 7. Spontaneous chemiluminescence and TBARS yields. Incubations consisted of cytochrome c‘+ (10 PM), 500 pM H202 (2.6 pmol lipid phosphate/ml), 10 mM potassium phosphate, pH 7.4, at 37°C. The bar graph represents TBARS produced after 30 min incubation. The lines show the time course of spontaneous chemiluminescence.

DISCUSSION Cytochrome c3+catalyzed peroxidation of PC liposomes by H202. More than one chemical mechanism accounted for cytochrome c plus H,O,-dependent lipid peroxidation, since there were both DTPA-inhibitable and DTPA-resistant components. This indicates that lipid peroxidation depended in part on free iron released from the hemeprotein. Iron release from 10 pM cytochrome c was greatly stimulated by Hz02 concentrations of 200 PM or higher, as judged by enhanced lipid peroxidation yields in the

I

1.0-j

TABLE

IV

Effect of Antioxidants on Cytochrome c3+Degradation and Liposome Lipid Peroxidation % Inhibition Reagent

”

KCN Urate DMSO Benzoate SOD

I

380

420

460 500 h (nm)

540

Cytochrome

c degradation 79 61 14 14 0

Lipid peroxidation 100 100 6 1 0

580

FIG. 6. Spectroscopic changes of cytochrome c3+ induced by H,O,. Cytochrome c3+ (10 PM) was incubated for 1 h with 100 pM HZ02 in 10 mM potassium phosphate, 20 j.tM DTPA, pH 7.4, at 37’C (a) minus H,O, or (b) plus H,On. Note a 5-nm shift to the left of the Soret absorbance peak in the H,O*-treated sample. The inset is a time scan of Adw for samples (a) and (b).

Cytochrome cl+, (10 FM) was incubated with 100 &M H202 and either 1 mM KCN, 1 mM urate, 1 mM DMSO, 1 mM benzoate, or 100 U/ml SOD. All reactions were in 10 mM potassium phosphate plus 20 pM DTPA, pH 7.4, at 37’C for 60 min. Cytochrome c degradation was measured by AA,,. In the absence of antioxidants, A,, decreased 28% from 0.9 to 0.65. For lipid peroxidation measurements, liposome concentration was 2.6 pmol lipid phosphate/ml. In the absence of antioxidants, TBARS were 0.8 WM.

CYTOCHROME

c AND

absence of DTPA (Fig. 1). Porphyrin rings can be oxidized and opened by Hz02 (34) leading to iron release when H202 concentrations are lo- to 30.fold greater than hemeprotein (28). The partial inhibition of lipid peroxidation by DTPA (Figs. 1 and 2) suggested that other species, in addition to free iron, were responsible for catalyzing HzO,-dependent lipid peroxidation. When free iron was added to catalyze H,Oz-dependent lipid peroxidation, DTPA was able to completely block this process. Free hemin or even fully coordinated iron mediation was ruled out, since hemin and potassium ferricyanide did not stimulate H202-dependent peroxidation of liposomes in the presence of DTPA. Thus, cytochrome c by itself appears to be mediating DTPA-resistant lipid peroxidation. Hydrogen peroxide can oxidize different organic molecules such as 4-aminoantipyrine, 2,2’-azino-bis(ethylbenzothiazoline sulfonic acid) and luminol in the presence of cytochrome c3t (24, 25). These molecules may bind to a hydrophobic pocket of cytochrome c and become oxidized by a high oxidation state oxo-iron complex (24). Ferrocytochrome c does not support oxidation of these molecules, first requiring oxidation to the ferric state (24). However, for oxidation of liposome membrane polyunsaturated acyl groups, there would be aqueous-hydrophobic phase incompatibility and steric restrictions, preventing direct interaction with the ferry1 or perferryl species. Cytochrome c can interact electrostatically with anionic membrane phospholipids, such as the cardiolipin of inner mitochondrial membranes (35) because it has eight positively charged residues at pH 7.4. Nevertheless, cytochrome c binding to liposome phospholipids was not a major requirement for H,Oz-dependent lipid peroxidation because of the minor influence of liposomal charge and ionic strength on peroxidation yields (Tables I and II). In contrast, cytochrome c-dependent auto-oxidation of the negatively charged liposomes occurred, supporting the concept that avid binding between hemeprot,ein and membrane was required in this process. In turn, hemin stimulation of liposome auto-oxidation was probably due to its hydrophobic properties, permitting interactions with the lipid bulk phase. Cytochrome c plus H,O, lipid peroxidation did not require identifiable protein-lipid binding and was insensitive to DTPA (Fig. 5), suggesting that iron was still heme-bound. Thus, it is likely that cytochrome c initiated peroxidation after partial protein oxidation and denaturation, so that reactive oxo-iron complexes became available for liposome reaction. The lag phase prior to lipid peroxidation further supports the concept of cytochrome c degradation or denaturation (DTPA noninhibitable component) followed by iron release (DTPA inhibitable component) as the sequence of events leading to enhancement of lipid peroxidation. The latter component of peroxidation predominated at concentrations greater than 200 pM (Fig. 1). Interestingly,

LIPID

123

PEROXIDATION

the loosely bound or “free” iron species could be readily transferred to and chelated by ATP, since the ATP-iron complex stimulated lipid peroxidation to a greater extent than free iron. Recent observations support the concept that most of the low molecular weight iron pool is present as ATP-iron (20). Iron derived from ferricytochrome c can induce lipid peroxidation through a mechanism that involves an initial oxidation of HzOz by ferric iron (Eq. [l]; Refs. (12, 36, 37)), thus initiating a radical chain reaction with participation of Haber-Weiss chemistry (Eqs. [2-31): AGO Fe”+ + H,O, + Fe’+ + 0; + 2H+ Fe’+ + H 20. L -+ Fe”+ + ‘OH + OHFe”’ + 0; + Fe” + 0, Fe”+ + 2H,02 + 2Fe’. + 0% + ‘OH + OH

[II [21 [31 + 2H’

[41

(kcal/mol) +18.9 --4.8 -10.4 +3.7.

Although Reaction 1 has a very positive standard free energy, it can still occur because initial concentrations of Fe’+ and 0; are zero and O,, once formed, will disappear rapidly by spontaneous dismutation. Moreover, considering reactions 1 to 3 together, there is a net Ae of only $3.7 kcal/mol (Eq. [4]) so that the exergonic nature of reactions 2 and 3 will favor the completion of reaction 1. Alternatively, reaction of H202 with ATP-Fe can yield high oxidation states of iron such as ferry1 iron (FeO”+) which has similar reactivity to ‘OH (14). Inhibitors of cytochrome c heme degradation such as cyanide and urate prevented lipid peroxidation (Table IV). Cyanide probably competed with H,O, for the sixth ligand position of the heme, as it does with other hemeproteins and as was previously suggested for cytochrome c (24,26). Uric acid protection from H,O,-dependent, cytochrome c degradation can be explained by preferential uric acid oxidation near the site of generation of reactive species or site(s) of reaction with cytochrome c3+. Uric acid has been proposed as a biological antioxidant and has a very low one-electron redox potential (38). The modest effects of DMSO and benzoate and the absence of effect of SOD show that ‘OH and 0, are not key intermediates. The TBARS produced during cytochrome c degradation can be attributed to heme degradation and secondarily to degradation of amino acids such as tryptophan, since open porphyrin derivatives such as bilirubin and oxidized amino acids react with TBA (39). Spontaneous chemiluminescence obtained from cytochrome c”+ exposure to H202 is consistent with previous reports, although we used substantially lower concentrations of H202 and thus could detect lag phases. There is disagreement on whether singlet molecular oxygen participates in these photochemical processes (26, 40), although it appears that most chemiluminescence depends on porphyrin ring reaction with the oxo-iron complex (40). Addition of liposomes caused a small but significant

124

RADI,

TURRENS.

increase in chemiluminescence (Fig. 7). Lipid peroxidation generates excited species such as singlet oxygen and excited carbonyls that decay to their ground states with emission of light (41, 42). Chemiluminescence in this reaction system did not directly reflect lipid peroxidation. The general assumption that TBARS formation and spontaneous chemiluminescence are analogous indicators of lipid peroxidation may be misleading, even in simple biochemical systems. When 500 FM H202 reacts in the presence of cytochrome c3+ and liposomes, 95% of TBARS formation was due to lipid peroxidation while 5% was due to protein oxidation (Fig. 7). This contrasts with the fact that 70% of chemiluminescence depends on protein oxidation and 30% on lipid peroxidation. It is possible though, that in the presence of liposomes, part of the reactive species generated during cytochrome c oxidation. If so, the contribution of lipid peroxidation to total chemiluminescence would be greater. Thus, these methods are not absolutely specific indicators of lipid peroxidation and TBARS formation and spontaneous chemiluminescence should be considered as complimentary rather than alternative approaches to studying lipid peroxidation in complex biological systems. Lipid peroxidation processes occur in mitochondrial membranes both in vitro and in viuo (43). Cytochrome P450, which is a component of rat liver mitochondrial inner membrane, initiates mitochondrial lipid peroxidation in the presence of cumene hydroperoxide and other hydroperoxides (44). Oxygen and ultraviolet light, ferrous iron, ascorbate, glutathione, Ccl, and chaotropic agents are mitochondrial peroxidizing agents as well (43). Peroxidation of the inner mitochondrial membrane causes inhibition of respiration, uncoupling of oxidative phosphorylation and swelling (43). We propose that reactions between cytochrome c and HZOZ could account, in part, for mitochondrial lipid peroxidation when there is an increased steady-state concentration of Hz02 in cells. Bovine heart mitochondria contain approximately 0.65 nmol cytochrome c/mg protein (45). With a matrix volume near 1.2 Qmg protein (75% of total mitochondrial volume), a concentration of about 400 PM cytochrome c for the whole mitochondria is predicted, with a much greater concentration predicted when considering only the intermembrane space (45). Hydrogen peroxide steady-state concentrations in mitochondria are expected to increase several times under some pathological situations (7-10) enhancing cytochrome c-catalyzed oxidative reactions. Cytochrome c-induced membrane peroxidation could preferentially affect specific phospholipids to which cytochrome c binds, such as cardiolipin (34). Concurrently, H,O, locally produced at high concentrations could trigger iron transfer from the high (i.e., cytochrome c) to the low (i.e., ATP) molecular weight pool, further increasing lipid peroxidation yields. We conclude that cytochrome cHTOz-induced lipid peroxidation should be considered representative of “site specific” lipid peroxidation reac-

AND

FREEMAN

tions applicable to other cytochromes which are intrinsic membrane proteins. ACKNOWLEDGMENTS We thank Kenneth M. Bush and Thomas P. Cosgrove for technical assistance. This work was supported by N.I.H. Grants ROl-HL40458 and ROl-NS24275 to B.A.F. and the Alabama Heart Association, Grant AL-G-880007 to J.F.T.

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3. Turrens,

J. F., Alexandre, A., and Lehninger, Biochem. Biophys. 217,401-410.

4. Miller,

D. Biol. Med. 5. Fridovich, Vol. 1, pp.

A. L. (1985) Arch.

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