Iron-mediated formation of an oxidized adriamycin free radical

Electron paramagnetic resonance studies are reported which demonstrate that the reduction of Fe3+ to Fe2+ by adriamycin results in the formation of an oxidized adriamycin free radical with an EPR signal at g = 2.004. A transient iron-adriamycin free radical complex is also observed at g = 2.34. The free radical is quantitated and its aerobic stability is determined. Observation of the oxidized adriamycin free radical signal confirms that adriamycin donates an electron to the bound Fe3+. In the presence of glutathione the drug-mediated reduction of Fe3+ to Fe2+ is bypassed, and the oxidized adriamycin radical signal is not observed. The oxidized adriamycin radicals and reduced oxygen radicals which are formed are two different mediators, whose relative concentrations could modulate the therapeutic and toxic effects of adriamycin.     

Hyperbaric Oxygen Therapy Increases Free Radical Levels in the Blood of Humans

It has been postulated that exposure to high concentrations of oxygen results in increased oxygen radical production which may account for the toxic effects of excessive exposure to oxygen. Examination of blood from persons undergoing hyperbaric oxygen (HBO) exposure, by low temperature electron spin resonance (ESR) spectroscopy, demonstrated a marked increase in the magnitude of a signal with properties consistent with a free radical (g = 2.006). The signal diminished to baseline levels within 10 minutes of cessation of HBO exposure. Further in vitro studies of blood revealed an ESR signal generated in red blood cells by oxygen, and dependent on oxyhaemoglobin, which had characteristics indistinguishable from those of the ESR signal of ascorbate radical and the signal in blood from persons undergoing HBO exposure. It is postulated that HBO exposure increases ascorbate radical levels in blood, which is likely to reflect increased ascorbate turnover in human red blood cells.     

Characterization of the free radical of mammalian ribonucleotide reductase

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.     

Metabolism of diethylstilbestrol by horseradish peroxidase and prostaglandin-H synthase. Generation of a free radical intermediate and its interaction with glutathione

Diethylstilbestrol is carcinogenic in rodents and in humans and its peroxidatic oxidation in utero has been associated with its carcinogenic activity. Horseradish peroxidase-catalyzed oxidation of [14C]diethylstilbestrol and [14C]diethylstilbestrol analogs induced binding of radiolabel to DNA only when the compound contained a free hydroxy group (Metzler, M., and Epe, B. (1984) Chem. Biol. Interact. 50, 351-360). We have found that horseradish peroxidase or prostaglandin-H synthase-catalyzed oxidation of diethylstilbestrol in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide caused the generation of an ESR signal indicative of a free radical intermediate (aN = 14.9 G, aH = 18.3 G). The identity of the trapped radical could not be identified on the basis of published hyperfine coupling constants, but the observation that horseradish peroxidase-catalyzed oxidation of 1-naphthol produced an identical ESR signal suggests that the radical was either a phenoxy or phenoxy-derived radical. During horseradish peroxidase-catalyzed oxidation of diethylstilbestrol in the presence of glutathione the thiol reduced the diethylstilbestrol radical to generate a thiyl radical. This was shown by a thiol-dependent oxygen uptake during horseradish peroxidase-catalyzed oxidation of diethylstilbestrol and the observation of an ESR signal consistent with 5,5-dimethylpyrroline-N-oxide-glutathionyl radical adduct formation. A diethylstilbestrol analog devoid of free hydroxy groups, namely diethylstilbestrol dipropionate, did not produce an ESR signal above control levels during horseradish peroxidase-catalyzed metabolism in the presence of 5,5-dimethylpyrroline-N-oxide. Thus, free radicals are formed during peroxidatic oxidation of diethylstilbestrol and must be considered as possible determinants of the genotoxic activity of this compound.     

Electron Spin Resonance Studies on Isolated Hepatocytes Treated with Ferrous Or Ferric Iron

Isolated rat hepatocytes incubated with iron salts in the presence of the spin trapping agent tx-4-pyridyl-l-oxide N-tert-butyl nitrone (4-POBN) generate a clear electron spin resonance signal; this signal is not detectable in the absence of exogenous iron. The hyperfine splitting constants are identical whether ferrous or ferric iron is used. The free radical trapped does not appear to be an active oxygen species but rather a carbon-centred radical, which we here ascribe to a lipodienyl radical on the basis of its hyperfine splitting features. Support to this interpretation is lent by the fact that no such radical could be generated in hepatocytes fully protected against lipid peroxidation by pretreating the donor rats with α-tocopherol.     

Pulsed EPR studies of doublet signal and singlet-like signal in oriented Ca2+-depleted PS II membranes: location of the doublet signal center in PS II

Doublet signal and singlet-like signal induced in Ca(2+)-depleted PS II were investigated by pulsed EPR in one-dimensionally oriented photosystem (PS) II membranes. The doublet signal showed marked angular dependent change in its spectrum in term of the applied magnetic field, indicating that the magnetic dipole-dipole interaction is mainly responsible for the doublet signal. The singlet-like signal also showed angular dependence, which was less pronounced than that of the doublet signal. The parameters of dipole and exchange interactions used to simulate the doublet signal indicate that the signal arises from a magnetically coupled organic radical pair. Angular dependence of the doublet signal indicates that the radius vector of the radical pair (r) and the normal of the thylakoid membrane is at an angle of 65 degrees. Pulsed ELDOR studies in the oriented membranes indicate that the vector (R) connecting the doublet-signal center with the Y(D)(*) radical and the plane of the thylakoid membrane are at an angle of 8 degrees. Furthermore, the angle between the projections of the R and r vectors on the plane of the thylakoid membrane was determined to be 64 degrees. The location of the doublet-signal species in PS II is discussed.     

ESR spin trapping of a carbon-centered free radical from agonist-stimulated human platelets

Several free radical intermediates formed during synthesis of prostaglandin H synthase (PGHS) catalyze the biosynthesis of prostaglandins from arachidonic acid (AA). We attempted to directly detect free radical intermediates of PGHS in cells. Studies were carried out using human platelets, which possess significant PGHS activity. Electron spin resonance (ESR) spectra showed a g = 2.005 signal radical, which was formed by the incubation of collagen, thrombin, AA, and a variety of peroxides with human platelets. The ESR spectra obtained using 5,5-dimethyl-1 pyrroline N-oxide (DMPO) and alpha-phenyl N-tert.-butylnitron (PBN) were typical of an immobilized nitroxide. Extensive Pronase digestion of both the DMPO and PBN adducts allowed us to deduce that it was a carbon-centered radical. The formation of this radical was inhibited by potassium cyanide and by desferroxamine. Peroxides stimulated formation of the g = 2.005 signal radical and inhibited platelet aggregation induced by AA. PGHS cosubstrates increased the intensity of the radical signal but inhibited platelet aggregation induced by AA. Both S-nitro-L-glutathione and reduced glutathione quenched the g = 2.005 radical but could not restore platelet aggregatory activity. These results suggest that the carbon-centered radical is a self-destructing free radical formed during peroxide-mediated deactivation of PGHS in human platelets.     

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O₂. The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~ 90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.     

Electron paramagnetic and electron nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase

We have collected electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectra from the hydrogen peroxide compound of yeast cytochrome c peroxidase, termed ES, employing EPR microwave frequencies of 9.6 and 11.6 GHz. We have measured and analyzed the temperature dependence of the spin-lattice relaxation rate (1/T1) of the paramagnetic center of ES over the temperature range 1.9 to 4 K. In addition, an upper bound to exchange coupling between the ferryl heme and EPR-visible centers of ES has been calculated and expressions for the dipolar interaction between a ferryl heme and a free radical have been derived. These results all confirm that the EPR signal of ES is not associated with an aromatic amino acid radical, and in particular not with a tryptophanyl radical. This conclusion has led us to consider an explanation of the EPR signal in terms of a nucleophilically stabilized methionyl radical.     

The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.     

Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide.

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water, a process which is accompanied by the pumping of four protons across the membrane. Elucidation of the structures of intermediates in these processes is crucial for understanding the mechanism of oxygen reduction. In the work presented here, the reaction of H(2)O(2) with the fully oxidized protein at pH 6.0 has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical based on comparison with other paramagnetic centers in COX was approximately 20%. Recent crystallographic data have shown that one of the Cu(B) ligands, His 276 (in the bacterial case), is cross-linked to Tyr 280 and that this cross-linked tyrosine is ideally positioned to participate in dioxygen activation. Here selectively deuterated tyrosine has been incorporated into the protein, and a drastic change in the line shape of the EPR signal observed above has been detected. This would suggest that the observed EPR signal does indeed arise from a tyrosine radical species. It would seem also quite possible that this radical is an intermediate in the mechanism of oxygen reduction.     

Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism

The EPR signal recorded in reaction medium containing L-lysine and methylglyoxal is supposed to come from the anion radical (semidione) of methylglyoxal and cation radical of methylglyoxal dialkylimine. These free radical inter-mediates might be formed as a result of electron transfer from dialkylimine to methylglyoxal. The EPR signal was observed in a nitrogen atmosphere, whereas only trace amounts of free radicals were registered under aerobic conditions. It has been established that the decay of methylglyoxal anion radical on aeration of the medium is inhibited by superoxide dismutase. Using the methods of EPR spectroscopy and lucigenin-dependent chemiluminescence, it has been shown that nonenzymatic generation of free radicals including superoxide anion radical takes place during the interaction of L-lysine with methylglyoxal — an intermediate of carbonyl stress — at different (including physiological) pH values. In the course of analogous reaction of L-lysine with malondialdehyde (the secondary product of the free radical derived oxidation of lipids), the formation of organic free radicals or superoxide radical was not observed.     

Electron paramagnetic resonance characterization of tyrosine radical, M+, in site-directed mutants of photosystem II(t).

Photosystem II contains two well-characterized tyrosine radicals, D(.) and Z(.). Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site and is essential for enzymatic function. On the other hand, D forms a stable radical with no known role in oxygen evolution. D(.) and Z(.) give rise to similar, but not identical, room temperature electron paramagnetic resonance (EPR) signals, which can be distinguished by their decay kinetics. A third room temperature EPR signal has also been observed in site-directed mutants in which a nonredox active amino acid is substituted at the D or Z site. This four-line EPR signal has been shown to have a tyrosine origin by isotopic labeling (Boerner and Barry, 1994, J. Biol. Chem. 269:134-137), but such an EPR signal has never before been observed from a tyrosyl radical. The radical giving rise to this third unique signal has been named M+. Here we provide kinetic evidence that this signal arises from a third redox active tyrosine, distinct from tyrosine D and Z, in the photosystem II reaction center. Isotopic labeling and EPR spectroscopy provide evidence that M is a covalently modified tyrosine.     

A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

The free radical formed during the hydroperoxide-mediated deactivation of ram seminal vesicles is hemoprotein-derived

Prostaglandin synthase is a multi-enzyme complex which catalyzes the oxygenation of arachidonic acid to the various prostaglandins. During the oxygenation, the enzyme is self-deactivated and, on the basis of ESR data, it has been proposed to form a self-destructive free radical. The free radical was suggested to form from the oxygen lost from prostaglandin G2 during its reduction to prostaglandin H2, and the destructive species was therefore thought to be an oxygen-centered free radical, tentatively identified as the hydroxy radical. We have reinvestigated this ESR signal (g = 2.005) and have concluded, with the aid of the known ESR parameters for the hydroxy and other oxygen-centered free radicals, that the free radical formed during the oxygenation is neither a hydroxy nor any known oxygen-centred radical. Prostaglandin synthase is thought to be a hemoprotein, so this unknown ESR signal was compared with the previously observed free radical formed by the reaction of H2O2 with methemoglobin. This comparison indicates that the free radical formed by the reaction of prostaglandin G2 with ram seminal vesicles is hemoprotein-derived and may be formed by the oxidation of an amino acid(s) located near the iron of the heme.     

A tyrosine-derived free radical in apogalactose oxidase

Oxidation of apogalactose oxidase with ferricyanide leads to the formation of a stable free radical exhibiting distinctive optical absorption and EPR spectral features. The radical is associated with absorption in both near-UV and near-IR spectral regions, and its EPR spectrum is characteristic of an aromatic free radical with gav = 2.005. Reconstitution of both the apoenzyme and the free radical-containing form with copper substantially restores both the absorption spectra and the catalytic activity of the active enzyme, indicating that the preparation of the radical species does not significantly damage the protein. The absence of a free radical EPR signal in reconstituted and activated galactose oxidase containing nearly stoichiometric copper suggests the radical is an active site species relating to the free radical-coupled copper site previously proposed for this enzyme. Isotopic labeling experiments demonstrate that the radical derives from a tyrosine residue. The distinctive spectra associated with this radical indicate an environment which is different from that associated with the tyrosyl phenoxyl sites in other free radical enzymes.     

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.     

An organic radical in the lysine 2,3-aminomutase reaction.

Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K. Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor. The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine. Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine. The radical signal is complex, having multiple hyperfine transitions. The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine. The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state. Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical. The results indicate that lysine radicals participate in the rearrangement mechanism.     

Reaction of bovine cytochrome c oxidase with hydrogen peroxide produces a tryptophan cation radical and a porphyrin cation radical

Oxidized bovine cytochrome c oxidase reacts with hydrogen peroxide to generate two electron paramagnetic resonance (EPR) free radical signals (Fabian, M., and Palmer, G. (1995) Biochemistry 34, 13802-13810). These radicals are associated with the binuclear center and give rise to two overlapped EPR signals, one signal being narrower in line width (DeltaHptp = 12 G) than the other (DeltaHptp = 45 G). We have used electron nuclear double resonance (ENDOR) spectrometry to identify the two different chemical species giving rise to these two EPR signals. Comparison of the ENDOR spectrum associated with the narrow signal with that of compound I of horseradish peroxidase (formed by reaction of that enzyme with hydrogen peroxide) demonstrates that the two species are virtually identical. The chemical species giving rise to the narrow signal is therefore identified as an exchange-coupled porphyrin cation radical similar to that formed in horseradish peroxidase compound I. Comparison of the ENDOR spectrum of compound ES (formed by the reaction of hydrogen peroxide with cytochrome c peroxidase) with that of the broad signal indicates that the chemical species giving rise to the broad EPR signal in cytochrome c oxidase is probably an exchange coupled tryptophan cation radical. This is substantiated using H(2)O/D(2)O solvent exchange experiments where the ENDOR difference spectrum of the broad EPR signal of cytochrome c oxidase shows a feature consistent with hyperfine coupling to the exchangeable N(1) proton of a tryptophan cation radical.