©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Radical Production from the Reaction of Cytochrome c with Organic Hydroperoxides
AN ESR SPIN TRAPPING INVESTIGATION (*)

David P. Barr (§) , Ronald P. Mason

From the (1) Laboratory of Molecular Biophysics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanism for the reaction of cytochrome c with t-butyl hydroperoxide and cumene hydroperoxide was investigated. ESR spin trapping studies using 5,5-dimethyl-1-pyrroline N-oxide were performed to demonstrate the presence of hydroperoxide-derived peroxyl, alkoxyl, and methyl radicals. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide concentrations was used to determine the relative contributions of each radical adduct to each composite ESR spectrum. From these analyses, it was concluded that the alkoxyl radical of the hydroperoxide was the initial radical produced, presumably by homolytic scission of the O-O bond by ferric cytochrome c. This was in contrast to a previous ESR study that proposed a heterolytic peroxidase-type mechanism for the reaction of cytochrome c with organic hydroperoxides. Methyl radicals were produced from the -scission of the alkoxyl radical. The peroxyl radicals are shown to be secondary products formed from the reaction of oxygen with the methyl radical to produce the methyl peroxyl radical. In separate experiments, visible absorption spectroscopy revealed that the heme center was destroyed during the reaction. Both the heme destruction and production of radical adducts were inhibited by cyanide, presumably due to the formation of a cyanoheme complex.


INTRODUCTION

Cytochrome c is an intermembrane mitochondrial heme protein that functions in electron transport by shuttling electrons from cytochrome c reductase to the cytochrome c oxidase complex. There is one protoporphyrin IX heme unit/molecule of cytochrome c, and the iron cycles between the ferric and ferrous oxidation states in the native form (1) . A histidine nitrogen provides the fifth ligand for the heme iron, while the sixth position is occupied by a sulfur ligand from methionine (2, 3, 4) . Thus, in the native form, cytochrome c is hexacoordinate. However, the methionine ligand is readily displaced by the presence of an alternate ligand or even subtle changes in temperature, pH, or ionic strength (3). It has been proposed that the loss of the methionine ligand results in the loss of the electron transport capability of cytochrome c(4) .

In addition to electron transport, it has been suggested for several decades that cytochrome c might be involved in the hydroperoxide-dependent initiation of free radical-mediated lipid peroxidation and protein damage (5, 6, 7, 8, 9, 10) . Recently, ESR spectroscopy has been used to demonstrate that several heme proteins, including cytochrome c, can react with organic hydroperoxides to produce free radicals (11, 12, 13, 14, 15) . Kalyanaraman et al.(11) reported the direct ESR detection of peroxyl radicals from the reaction of hematin with t-butyl hydroperoxide and cumene hydroperoxide. In separate studies (13, 14) , the ESR spin trapping technique was employed to demonstrate that peroxyl, alkoxyl, and carbon-centered radicals are produced from the reaction of various heme proteins (including cytochrome c) with organic hydroperoxides. In such studies, a spin trap such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)() is added to a reaction mixture containing the heme protein and hydroperoxide. The resulting ESR spectrum reveals a mixture of radical adducts that can be identified by their characteristic nitrogen and hydrogen hyperfine coupling constants.

Based on ESR spectra and other data, several mechanisms have been postulated for radical production involving the reaction of heme proteins with organic hydroperoxides. A peroxidase-type mechanism is one possibility (11, 14) . In this case, the ferric form of cytochrome c would heterolytically cleave the peroxide O-O bond, and the organic hydroperoxide would be reduced to its corresponding alcohol. Also, a ferryl radical form of cytochrome c (i.e. compound I) would result that could abstract a hydrogen from another molecule of hydroperoxide, thus producing a peroxyl radical. Alternatively, the ferric cytochrome c could cleave the peroxide O-O bond homolytically, which would produce an alkoxyl radical. Tappel (16) has suggested that this might occur without a valence change of the heme iron. Instead, an iron-bound hydroxyl radical may be formed (16) . Yet another explanation invokes a cytochrome c-catalyzed Haber-Weiss reaction, in which the organic hydroperoxide replaces hydrogen peroxide (17, 18, 19) .

It has been concluded from recent ESR investigations that production of radicals by the cytochrome c/organic hydroperoxide system occurs via the peroxidase-type mechanism (13) . The results reported here do not support these previous conclusions. Instead, we have provided ESR spin trapping evidence that indicates that the alkoxyl radical is the primary radical released into solution by the reaction of cytochrome c with an organic hydroperoxide. The implications of this finding with regard to the aforementioned mechanisms and the free radical-mediated lipid peroxidation and protein damage of the cytochrome c/organic hydroperoxide system are discussed.


EXPERIMENTAL PROCEDURES

Chemicals

Horse heart cytochrome c was purchased from Boehringer Mannheim and used as received. DMPO, DTPA, EDTA, t-butyl hydroperoxide (t-BuOOH), and Desferal (desferrioxamine mesylate) were all obtained from Sigma. DMPO was vacuum-distilled at room temperature twice before use. Potassium cyanide and cumene hydroperoxide were obtained from Aldrich. Cumene hydroperoxide was purified by preparing the sodium salt according to the method of Mair and Hall (20) . The buffers and reagents were prepared in deionized water and were treated with Chelex 100 (Bio-Rad).

ESR Spin Trapping Experiments

The ESR spectra were recorded using a Bruker ECS-106 spectrometer operating at 9.77 GHz with a 50-kHz modulation frequency and a TM cavity. In the experiments shown in Fig. 1-4, the reactions were initiated by the addition of the hydroperoxide and pipetted into the flat cell. The time delay before the spectra were recorded was 1.5 min. In the experiments shown in Fig. 6and Fig. 7, the reaction mixtures were introduced to the flat cell by means of aspiration (with a dead time of only 4 s) using a modified Gilford rapid sampling device (21) . This was done to minimize the possible decay of radical adducts during the DMPO-dependence studies. All ESR experiments were performed in 100 mM sodium phosphate, pH 7.4, at room temperature. The reactions were initiated by the addition of the hydroperoxide. For the anaerobic experiments shown in Fig. 8, the oxygen was removed by nitrogen purging, as described previously (21) , using a bottle equipped with a rubber septum and syringe needles. The solution was purged for 10 min, after which glucose (10 mM) and glucose oxidase (100 units) were added. The reactions were initiated by the simultaneous addition of cytochrome c and DMPO. In both the aerobic and anaerobic experiments shown in Fig. 8, the mixture was introduced to the flat cell using the rapid sampling device.


Figure 1: Computer simulation and deconvolution of the ESR spectrum obtained from the reaction mixture containing cytochrome c and t-BuOOH. A is the computer simulation (dashedline) superimposed on the experimental spectrum obtained using 20 µM cytochrome c (Cytc), 12.5 mMt-butyl hydroperoxide, and 22.5 mM DMPO. B-E are the individual simulations of each species in the composite spectrum. The hyperfine values used for each species are provided under ``Results.'' B is the simulated spectrum for DMPO/OOR: line width, 0.70 G; line shape, 44% lorentzian, 56% gaussian; and mole ratio, 0.70. C is the simulated spectrum for DMPO/OC(CH): line width, 0.75 G; line shape, 42% lorentzian, 58% gaussian; and mole ratio, 0.22. D is the simulated spectrum for DMPO/CH: line width, 0.70 G; line shape, 18% lorentzian, 82% gaussian; and mole ratio, 0.04. E is the simulated spectrum for DMPOX: line width, 0.45 G; line shape, 49% lorentzian, 56% gaussian; and mole ratio, 0.04.




Figure 6: Effect of the DMPO concentration on the relative contributions of each radical adduct to the composite ESR spectrum obtained from the reaction of cytochrome c with t-butyl hydroperoxide. All reaction mixtures contained 20 µM cytochrome c, 12.5 mMt-butyl hydroperoxide, and 100 mM phosphate buffer, pH 7.4. The DMPO concentrations used were 22.5, 45, 112, 135, 158, 180, and 360 mM. , DMPO/OOR; , DMPO/OC(CH); , DMPO/CH; , DMPOX.




Figure 7: Effect of the DMPO concentration on the relative contributions of each radical adduct to the composite ESR spectrum obtained from the reaction of cytochrome c with cumene hydroperoxide. All reaction mixtures contained 20 µM cytochrome c, 25 mM cumene hydroperoxide, and 100 mM phosphate buffer, pH 7.4. The DMPO concentrations used were 45, 90, 135, 158, 180, 270, 360, and 720 mM. , DMPO/OOR; , DMPO/OC(CH)CH; , DMPO/CH; , DMPOX.




Figure 8: Effect of oxygen on the production of radical adducts from the reaction of cytochrome c with t-BuOOH and cumene hydroperoxide. A is the spectrum obtained in the presence of oxygen from a reaction mixture containing 20 µM cytochrome c, 25 mM cumene hydroperoxide (CumOOH), and 20 mM DMPO (CumOOH/Air). B is the spectrum obtained when the system was purged of oxygen using nitrogen bubbling and using the conditions from A (CumOOH/N). C is the spectrum obtained in the presence of oxygen from a reaction mixture containing 20 µM cytochrome c, 12.5 mMt-butyl hydroperoxide, and 20 mM DMPO (t-BuOOH/Air). D is the spectrum obtained when the system was purged of oxygen using nitrogen bubbling and using the conditions from C (t-BuOOH/N). The spectrometer conditions were the same as indicated in the legend of Fig. 2.



Computer Simulations

The computer simulations were performed using a program developed by David Duling at NIEHS that is available to the public through Internet. The details of this program were the subject of a recent publication (22) . The parameters that were used for simulating the experimental ESR spectra are described in the figure legends. The data points in Fig. 6and Fig. 7 were obtained by simulating the low-field lines of each species in spectra that were obtained using rapid sampling. The component spectra were then double-integrated to give relative radical adduct concentrations. Experiments using each DMPO concentration were performed both in the presence and absence of cytochrome c. Prior to computer simulation, the spectra obtained in the absence of cytochrome c were subtracted from those obtained in its presence. Thus, the contribution of the cytochrome c-independent species was eliminated.

Visible Absorption Spectroscopy

The spectral changes of cytochrome c were recorded using a multichannel photodiode on a Hewlett-Packard 8415A diode array spectrophotometer. The reactions were initiated by the addition of the hydroperoxide and were performed in 100 mM sodium phosphate, pH 7.4, at room temperature. Further details of these experiments are provided in the figure legends.


RESULTS

The ESR spectrum recorded from a reaction mixture containing cytochrome c, t-butyl hydroperoxide, and DMPO revealed a mixture of four DMPO radical adducts (Fig. 1) and was similar to what has been reported previously (13) . The computer simulation of the spectrum obtained using 22.5 mM DMPO was superimposed on the experimental spectrum (Fig. 1A). From their respective hyperfine coupling constants, the adducts were assigned as peroxyl (methyl peroxyl; see below) (a = 14.45 G, a = 10.75 G, and a = 1.35 G), t-butyloxyl (a = 14.9 G and a = 16.2 G), methyl (a = 16.4 G and a = 23.4 G), and 5,5-dimethylpyrrolidone-2-(oxy)-(1) (DMPOX) (a = 7.25 G and a(2H) = 4.05 G). The hyperfine values appeared to be consistent with the values reported in previous literature (14) . DMPOX is an oxidation product of DMPO that is thought to arise from the decomposition of DMPO peroxyl radical adducts (17) . Deconvolution of the composite spectrum was performed by simulating each species separately using the relative intensities and hyperfine values obtained from the composite computer simulation (Fig. 1, B-E). Inclusion of the DMPO hydroxyl radical adduct in the composite simulation did not improve the quality of the fit (i.e. the correlation coefficient did not increase). In addition, if t-butyl hydroperoxide was replaced by hydrogen peroxide, only DMPOX, not the DMPO hydroxyl radical adduct, was detected.

The relative contributions of the peroxyl and alkoxyl adducts to the composite ESR spectrum were influenced by the concentration of DMPO (Fig. 2, A, C, and E). Qualitatively, one can see that as the DMPO concentration was increased, the relative contribution of the peroxyl radical adduct decreased, while that of the t-butyloxyl radical increased. In the absence of cytochrome c, a composite spectrum with greatly reduced intensity was observed (Fig. 2, B, D, and F). The results were similar when cumene hydroperoxide replaced t-butyl hydroperoxide in the DMPO spin trapping experiments (Fig. 3). The computer simulation fit well with the experimental data (Fig. 3A), and the presence of each radical adduct can clearly be seen in the spectral deconvolution (Fig. 3, B-E). In the case of cumene hydroperoxide, the DMPO adducts were assigned as peroxyl (methyl peroxyl; see below) (a = 14.45 G, a = 10.70 G, and a = 1.35 G), cumyloxyl (a = 14.75 G and a = 15.84 G), methyl (a = 16.5 G and a = 23.35 G), and DMPOX (a = 7.25 G and a(2H) = 4.05 G). These hyperfine coupling values were similar to those reported by Davies (13) . Once again, as DMPO was increased, the contribution of the peroxyl radical adduct to the composite spectrum was diminished, while the cumyloxyl radical adduct contribution increased (Fig. 4, A, C, and E). The control experiments in the absence of cytochrome c are shown at each DMPO concentration (Fig. 4, B, D, and F).


Figure 2: Composite ESR spectrum of DMPO radical adducts from incubations of cytochrome c and t-BuOOH at various DMPO concentrations. A is the ESR signal obtained from a reaction mixture containing 20 µM cytochrome c (Cytc), 12.5 mMt-butyl hydroperoxide, and 22.5 mM DMPO (i.e. the same as seen in Fig. 1A). B is the spectrum obtained using the conditions from A, except cytochrome c was omitted. C is the spectrum obtained when the DMPO concentration was increased to 90 mM. D is the spectrum obtained using the conditions from C, except cytochrome c was omitted. E is the spectrum obtained when the DMPO concentration was increased to 360 mM. F is the spectrum obtained using the conditions from E, except cytochrome c was omitted. The spectrometer conditions were as follows: modulation amplitude, 0.5 G; microwave power, 20 milliwatts; time constant, 0.69 s; scan time, 600 s; scan range, 100 G; and receiver gain, 2.5 10 for A-D and 1.6 10 for E and F.




Figure 3: Computer simulation and deconvolution of the ESR spectrum obtained from the reaction mixture containing cytochrome c and cumene hydroperoxide. A is the computer simulation (dashedline) superimposed on the experimental spectrum obtained using 20 µM cytochrome c (Cytc), 25 mM cumene hydroperoxide (CumOOH), and 45 mM DMPO. B-E are the individual simulations of each species in the composite spectrum. The hyperfine values used for each species are provided under ``Results.'' B is the simulated spectrum for DMPO/OOR: line width, 0.70 G; line shape, 51% lorentzian, 49% gaussian; and mole ratio, 0.68. C is the simulated spectrum for DMPO/OC(CH)CH: line width, 0.75 G; line shape, 0% lorentzian, 100% gaussian; and mole ratio, 0.13. D is the simulated spectrum for DMPO/CH: line width, 0.75 G; line shape, 53% lorentzian, 47% gaussian; and mole ratio, 0.06. E is the simulated spectrum for DMPOX: line width, 0.45 G; line shape, 95% lorentzian, 5% gaussian; and mole ratio, 0.13.




Figure 4: Composite ESR spectrum of DMPO radical adducts from incubations of cytochrome c and cumene hydroperoxide at various DMPO concentrations. A is the spectrum obtained from a reaction mixture containing 20 µM cytochrome c (Cytc), 25 mM cumene hydroperoxide (CumOOH), and 45 mM DMPO (i.e. the same as seen in Fig. 3A). B is the spectrum obtained using the conditions from A, except cytochrome c was omitted. C is the spectrum obtained when DMPO was increased to 135 mM. D is the spectrum obtained using the conditions from C, except cytochrome c was omitted. E is the spectrum obtained when the DMPO concentration was increased to 360 mM. F is the spectrum obtained using the conditions from E, except cytochrome c was omitted. The spectrometer conditions were as follows: modulation amplitude, 0.5 G; microwave power, 20 milliwatts; time constant, 0.69 s; scan time, 600 s; scan range, 100 G; and receiver gain, 2.5 10 for A-D and 1.6 10 for E and F.



The origin of the signals observed in the absence of cytochrome c is unknown. The reagents used in these experiments were Chelex-treated to minimize the contribution of trace transition metals. In addition, neither EDTA nor DTPA had any effect on the concentrations of radical adducts in the cytochrome c-catalyzed reaction or the control reaction mixtures (data not shown). Desferal did decrease the intensity of the signal seen in the absence of cytochrome c (data not shown). However, the addition of Desferal to the cytochrome c-catalyzed reaction resulted in the scavenging of the hydroperoxide-derived radicals and the appearance of the Desferal nitroxide free radical (Fig. 5). The 1-electron oxidation of Desferal to a stable nitroxide has been demonstrated in several free radical-generating systems (23) . The hyperfine coupling constants used to simulate the spectra shown in Fig. 5B (dashedline) were obtained from a report by Morehouse et al.(23) and are as follows: a = 7.85 G and a(2H) = 6.35 G. Twenty-seven percent of the free radicals in the composite spectrum (Fig. 5B) were due to the Desferal nitroxide radical. Although Desferal is often used in these systems as a transition metal chelator (24, 25) , it is also a powerful radical scavenger and is even capable of reacting with superoxide (23) . Thus, it is unclear whether Desferal was acting as a metal chelator or a radical scavenger. The addition of 10 mM cyanide to the cytochrome c-catalyzed reaction also decreased the radical adduct concentrations to the levels seen in the control experiments, giving a spectrum that was predominantly the DMPO hydroxyl radical adduct (data not shown). Cyanide coordinates with the heme iron of cytochrome c at the sixth axial position (26, 27) and thereby prevents its reaction with the hydroperoxide.


Figure 5: Effect of Desferal on the production of radical adducts from the reaction of cytochrome c with t-BuOOH. A is the spectrum obtained from a reaction mixture containing 20 µM cytochrome c (Cytc), 12.5 mMt-butyl hydroperoxide, and 90 mM DMPO. B is the spectrum obtained using the conditions from A, except Desferal (50 µM) was added. The dashedline superimposed on the experimental spectrum in B is the computer simulation. The hyperfine values used for the simulation are provided under ``Results.'' The radical adducts included in the simulation were as follows. DMPO/OOR: line width, 0.75 G; line shape, 58% lorentzian, 42% gaussian; and mole ratio, 0.40. DMPO/OC(CH): line width, 0.65 G; line shape, 0% lorentzian, 100% gaussian; and mole ratio, 0.31. DMPO/CH: line width, 0.65 G; line shape, 0% lorentzian, 100% gaussian; and mole ratio, 0.02. Desferal nitroxide: line width, 0.65 G; line shape, 99% lorentzian, 1% gaussian; and mole ratio, 0.27. The spectrometer conditions were the same as indicated in the legend of Fig. 2.



The data in Fig. 2and Fig. 4made it apparent that the proportions of the radical adducts observed in the composite spectrum were highly dependent on the concentration of DMPO. As will be discussed later, there are a number of reactions by which alkoxyl radicals can be produced from peroxyl radicals and vice versa. However, the different mechanisms proposed for the reaction of cytochrome c with these hydroperoxides dictate that the initial radical produced is either the peroxyl radical (as predicted by the heterolytic peroxidase mechanism) or the alkoxyl radical (via the homolytic mechanism).

The reactions of the initial radical that occur subsequent to its production can be suppressed by increasing the concentration of the spin trap. Thus, as more spin trap is added, more of the initial radical is trapped. This prevents the initial radical from undergoing reactions with other species or itself that lead to secondary radicals. By doing this, one can determine which radical is the primary or initial radical in a multiple species ESR spectrum. This method was used previously by Ledwith et al.(28) . We used this strategy to determine whether the peroxyl or the alkoxyl radical was the initial radical produced by the reaction of cytochrome c with these organic hydroperoxides. To do this, the relative contributions of the DMPO adducts were measured at various DMPO concentrations. In these experiments, the relative concentrations of the radical adducts as represented by the low-field lines were calculated using computer simulation. To eliminate the possible contribution of non-cytochrome c-catalyzed radical production (e.g. seen in Fig. 2, B, D, and F), parallel experiments were performed in the absence of cytochrome c at each DMPO concentration. Spectral subtractions were then performed, thus removing the contribution of cytochrome c-independent radical production. It can clearly be seen that as the DMPO concentration was increased between 22.5 and 360 mM, the contribution of the peroxyl radical to the composite ESR spectrum decreased significantly (i.e. from 85 to 15%) (Fig. 6). The t-butyloxyl radical, on the other hand, constituted only 10% of the composite spectrum at 22.5 mM DMPO. However, at 360 mM DMPO, the t-butyloxyl radical accounted for 80% of the radicals that were trapped. The contribution of the methyl radical, which arises from the -scission of the t-butyloxyl radical, and the DMPOX adduct remained nearly unchanged as the DMPO concentration was varied.

The same experiment was performed with cumene hydroperoxide, and a similar result was found (Fig. 7). As the DMPO concentration was increased between 45 mM and 720 mM, the peroxyl radical adduct decreased from 86 to 0%, while the cumyloxyl radical adduct increased from 10 to >90%. The contributions of the methyl radical and DMPOX adducts remained minimal (i.e. <10%) throughout the range of DMPO concentrations.

The peroxyl radical adduct detected in the preceding experiments could have arisen from one or all of the following reactions.

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Because of the spectroscopic ambiguity in the ESR of peroxyl radical adducts, it is not possible to differentiate between the methyl peroxyl radical adduct and tertiary peroxyl radical adducts. Thus, Reactions 1-3 are all valid possibilities. To determine if the peroxyl radical was being produced by Reaction 1, experiments were performed in the absence of oxygen (Fig. 8). A low DMPO concentration was used to maximize the intensity of the peroxyl radical adduct. It can be seen with both hydroperoxides that when oxygen was removed from the system, the signal intensity of the peroxyl radical adduct diminished almost completely. In addition, the intensity of the methyl radical adduct increased significantly. It is possible to detect tertiary peroxyl radicals using direct ESR (11) . However, under our experimental conditions, peroxyl radicals were not detected via direct ESR.

We also studied the spectral changes that occurred in the Soret and visible absorption bands of cytochrome c during its reaction with t-butyl hydroperoxide and cumene hydroperoxide. Ferricytochrome c has a maximum Soret absorbance at 410 nm as well as another maximum absorbance at 528 nm (29). When cytochrome c is converted to the ferrous form, the Soret band is slightly red-shifted (i.e. 416 nm), and prominent - and -absorption bands appear at 550 and 521 nm, respectively (29) . In addition, both the ferric and ferrous forms of native cytochrome c have a charge transfer absorption maximum at 695 nm that is due to the iron-sulfur bond of the axial methionine ligand (3) . This 695 nm band has been used as an indicator that cytochrome c is in its native hexacoordinate form (3, 4) . It has been reported previously that the Soret and visible bands of ferricytochrome c are bleached upon the addition of hydrogen peroxide and t-butyl hydroperoxide (7, 18) . Also, the characteristic red shift of the Soret band associated with compound I formation when a ferriperoxidase reacts with HO was not observed for cytochrome c(18) . Based on these results, it has been previously concluded that a compound I form of cytochrome c is not formed from the reaction with hydroperoxides. It has also been suggested that the heme iron of cytochrome c cannot coordinate with the peroxide oxygen because it is already hexacoordinate (18) . However, separate studies using the 695 nm absorption band as well as proton NMR have shown that even minor perturbations in pH, temperature, or ionic strength result in the loss of the sixth methionine ligand (3, 4) . We found that the addition of either t-butyl or cumene hydroperoxide to cytochrome c (data not shown for t-butyl hydroperoxide) resulted in the rapid bleaching of the Soret band as well as the 528 nm absorbance maximum (Fig. 9A). The - and -absorption bands characteristic of the ferrous form of cytochrome c were not observed at any time during the reaction. The 695 nm band was observed prior to the addition of the hydroperoxides (which indicated that the cytochrome c was initially hexacoordinate under our experimental conditions), but disappeared with time after the hydroperoxides were added (data not shown). Consistent with the ESR data, the addition of cyanide prevented the bleaching (Fig. 9B). Instead, a cytochrome c-cyanide complex was observed that was similar to that demonstrated in previous reports (26, 27) . On the other hand, the addition of DMPO did not affect the bleaching when either t-butyl or cumene hydroperoxide was added (data not shown).


Figure 9: Visible absorption spectra of cytochrome c following the addition of cumene hydroperoxide. In A, the reaction mixture contained 20 µM cytochrome c and 25 mM cumene hydroperoxide. B was obtained using the conditions from A, except 10 mM cyanide was added just prior to the addition of cumene hydroperoxide. Absorption spectra were recorded every 45 s. The arrows indicate the initial scan and the direction of the absorbance change with time.




DISCUSSION

The mechanism by which radicals are produced from the reaction of organic hydroperoxides with heme proteins has been the subject of considerable debate. The complexity in interpreting these data arises from the fact that a multitude of reactions can occur subsequent to the reaction of the heme iron with the hydroperoxide. For example, the peroxidase mechanism predicts that the peroxyl radical would be produced by the reaction of t-butyl hydroperoxide with cytochrome c (Cytc).

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Thus, in this case, the hydroperoxide would undergo a 2-electron reduction, in a heterolytic manner, to its corresponding alcohol, and the cytochrome c would be oxidized to a compound I-type species (Reaction 4). The resulting compound I could then abstract hydrogen from the organic hydroperoxide, producing a peroxyl radical and the ferryl form of cytochrome c (Reaction 5). The well established self-reaction of tertiary peroxyl radicals (30, 31) would result in the formation of two alkoxyl radicals and molecular oxygen (Reaction 6). Subsequent unimolecular -scission of the alkoxyl radical would form the methyl radical (Reaction 7) (32) . Therefore, this mechanism can account for all the radicals that are observed in the spin trapping studies. In fact, Davies (13) invoked the peroxidase mechanism to explain his ESR results from the reaction of organic hydroperoxides with various heme proteins including cytochrome c. However, the peroxidase mechanism would dictate that the peroxyl radical be produced initially and that the alkoxyl and methyl radicals be formed as products of its breakdown. If this were true, one would expect that as the DMPO concentration was increased, more peroxyl radical would be trapped and less of the DMPO alkoxyl radical adduct would be detected. In fact, the exact opposite was observed in the present study. It was clearly demonstrated that the alkoxyl radical adduct dominated at the highest DMPO concentrations. In addition, the peroxyl radical adduct decreased to a very nominal proportion of the total radical adduct as the concentration of spin trap was increased. Using the plots shown in Fig. 6 and Fig. 7, one could extrapolate that as the DMPO concentration was raised infinitely, the percent of the ESR spectrum resulting from the peroxyl radical adduct would approach zero, while that of the alkoxyl radical adduct would approach 100.

To explain how all three of these radical adducts were observed in the ESR spin trapping experiments, we have considered the following reaction sequence (t-butyl hydroperoxide has been used for demonstration here, but an analogous reaction sequence could be inferred for cumene hydroperoxide).

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In this reaction sequence, cytochrome c would catalyze the homolytic scission of the peroxide O-O bond (Reaction 8), possibly forming a ferryl form of cytochrome c. The -scission of the tertiary alkoxyl radical (Reaction 9) would yield the methyl radical that was observed. The reaction of the methyl radical with oxygen results in the formation of the methyl peroxyl radical (Reaction 10). The peroxyl radical adduct signals observed were primarily due to the methyl peroxyl radical. Evidence for this conclusion was obtained when oxygen was omitted from the reaction mixture. Under anaerobic conditions, the peroxyl radical adduct signal disappeared and was almost completely replaced by the methyl radical adduct signal. It should be noted that the hyperfine coupling constants for the DMPO methyloxyl radical adduct are typical of DMPO peroxyl radical adducts (33). Thus, the signals we are assigning to the methyl peroxyl adduct might be due to the methyloxyl radical adduct. In either case, the radical adduct would be derived from the methyl radical and thus would not change our mechanistic conclusions (i.e. Reactions 8-10).

The spin trapping reaction,

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(which has a rate constant of 4 10M s) (12) competes with -scission. Therefore, as more alkoxyl radical is trapped (i.e. more spin trap is added), less peroxyl radical can be formed via Reactions 9 and 10. The DMPOX observed in either case is probably due to the breakdown of the peroxyl radical and/or the oxidation of DMPO by the ferryl species. The methyl radical adduct remained relatively unchanged as the DMPO concentration was increased. Apparently, as DMPO was increased, the rate of the methyl radical trapping increased to compensate for the decrease in methyl radical production via the -scission Reaction 7. Thus, even though less methyl radical was being formed due to the increased trapping of t-BuO (Reaction 11), the amount of methyl radical adduct detected remained approximately the same.

Cadenas et al.(7) demonstrated singlet oxygen production from the reaction of cytochrome c with organic hydroperoxides. They concluded that cytochrome c catalyzed the homolytic scission of the hydroperoxide O-O bond and proposed that the chemiluminescence observed was due to the self-terminating reaction of primary peroxyl radicals that were derived from t-butyl alkoxyl radicals. It is likely that the methyl peroxyl radical would undergo a self-terminating reaction, producing singlet oxygen.

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The problem with making a mechanistic conclusion based only on chemiluminescence data is that the methyl peroxyl radicals could also have been derived from the peroxidase mechanism (i.e. Reactions 4-7). However, the spin trapping data presented here seem to rule out the possibility that the peroxidase mechanism is occurring with cytochrome c.

Another possible mechanism put forth for the reaction of cytochrome c with hydroperoxides involves a Haber-Weiss-type reaction sequence (17, 18, 19) .

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This implies that the hydroperoxide is first oxidized by 1 electron to the peroxyl radical, with the concomitant reduction of cytochrome c. Ferrous cytochrome c can then react with another molecule of hydroperoxide in a Fenton-type reaction (i.e. Reaction 14). Once again, Reactions 13 and 14 would dictate that the peroxyl radical be produced initially, followed by the formation of the alkoxyl radical. Thus, if Reactions 13 and 14 had occurred, one would predict that as the DMPO concentration was increased, both the peroxyl and alkoxyl radical adducts would have increased simultaneously (which was not the case). In addition, no spectrophotometric evidence for ferrous cytochrome c was obtained. Therefore, we concluded that radical production via Reactions 13 and 14 was unlikely. It has also been reported that iron release from cytochrome c might be the cause for radical production (18). However, the absence of an effect of EDTA or DTPA on the observed ESR signals in our cytochrome c/hydroperoxide reaction systems seemed to rule out this possibility. Also, the addition of cyanide reduced the intensity of the ESR signals to the level seen in the control experiments. This suggested that radical production required the interaction of the heme iron with the hydroperoxide.

The question as to the fate of the ferryl cytochrome c (which was used to balance Reaction 8) remains unanswered. One might expect that it would abstract a hydrogen from the hydroperoxide to produce a peroxyl radical and ferric cytochrome c.

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On-line formulae not verified for accuracy

In this case, the peroxyl and alkoxyl radical adducts would have increased simultaneously as the concentration of DMPO was increased, which was not observed. It is possible that the bleaching of cytochrome c observed during the reaction was due to an attack of the ferryl iron on the protein. The fact that cyanide inhibited the bleaching indicated that an interaction of the hydroperoxide with the heme iron (i.e. inner sphere electron transfer) was required for protein damage to occur. In addition, the fact that DMPO did not prevent bleaching suggested that the protein damage was not caused by diffusible hydroperoxide-derived radicals. However, more experimental data are required to address this question.

We believe that the ESR data presented here provide strong evidence for the homolytic scission of the O-O bond by cytochrome c initially producing alkoxyl radicals. The peroxyl radicals detected in these as well as previous ESR studies likely arise from Reaction 1 and, possibly, Reactions 2 and 3. The results have relevance with regard to heme protein-catalyzed lipid peroxidation and/or protein damage. Both peroxyl and alkoxyl radicals are capable of abstracting allylic hydrogens and thus of initiating lipid peroxidation. However, allylic hydrogen abstraction by alkoxyl radicals is more favorable. This is because the bond energy for the corresponding alcohol produced from hydrogen abstraction by an alkoxyl radical (104 kcal/mol) is considerably higher than that of the hydroperoxide resulting from hydrogen abstraction by a peroxyl radical (90 kcal/mol) (34) . In fact, pulse radiolysis studies have shown that the t-butyloxyl radical abstracts hydrogens from several polyunsaturated fatty acids at a rate of 2.0 10M s(35) , while the rate constant for hydrogen abstraction by peroxyl radicals is only 5 10M s (31). Thus, the finding that alkoxyl radicals are formed initially in the cytochrome c-catalyzed degradation of hydroperoxides may have significance when considering the relative roles of alkoxyl and peroxyl radicals in heme protein-catalyzed lipid peroxidation and/or protein damage.


FOOTNOTES

*
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§
To whom correspondence should be addressed. Tel.: 919-541-7573; Fax: 919-541-7880.

The abbreviations used are: DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DMPOX, 5,5-dimethylpyrrolidone-2-(oxy)-(1); DTPA, diethylenetriaminepentaacetic acid; t-BuOOH, t-butyl hydroperoxide.


ACKNOWLEDGEMENTS

We thank Dr. Krzysztof Reszka for helpful discussions and for suggesting the anaerobic experiments.


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