Semiquinone anion radicals of catechol(amine)s, catechol estrogens, and their metal ion complexes.

The characterization and identification of semiquinone radicals from catechol(amine)s and catechol estrogens by electron spin resonance spectroscopy is addressed. The use of diamagnetic metal ions, especially Mg2+ and Zn2+ ions, to detect transient semiquinone radicals in biological systems and to monitor their reactions, is discussed. A brief account of the identification and reactions of quinones is also presented.

This review addresses the generation, identification, and reactions of o-semiquinones and o-quinones from catechol(amine)s and catechol estrogens in a biochemical milieu.
Inactivation of enzymes/proteins by o-quinones is presumably due to the nucleophilic addition reactions (of quinones) to sulfhydryl or amino groups present in these macromolecules (16,58). The production of 5-S-cysteinyl dopa in the urine of melanoma patients (64)  isolation of quinone-alanine adduct(s) in terrestrial humic acid (65) provide indirect evidence for occurrence of these addition reactions. Reactions between o-quinones and proteins provide a basis for formation of "melano-proteins" (66). The toxicity of adrenochrome (a material which leads to myocardial necrosis) has been attributed to formation of the (one-electron) reduced free radical and to the oxy-radicals derived from it (via redox cycling of oxygen) (67-69).

Photooxidation
Photooxidation provides a simple, clean method for generating semiquinone radicals and studying their reactions. The primary photoreaction in dopa, catechol, and a variety of other catecholamines involves a mixture of photoionization and photohomolysis (81). This was established by a quantitative spin trapping procedure that we developed using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which scavenges both hydrated electrons and hydrogen atoms formed during the photooxidation.
The quantum yield for semiquinone formation is ca. 0.04 at pH 7, increasing to about 0.08 at higher pH where the catechol moiety is ionized. Sensitized photolysis of catechols and catecholamines has been demonstrated using a variety of dyes. Visible irradiation in the presence of hematoporphyrin, Rose Bengal, methylene blue, and other sensitizers gives intense spectra of corresponding semiquinones (70).
In general, photooxidation (using a slow flow) can be used for (1) generating specific primary radicals for characterization purposes without interference from secondary radicals and (2) obtaining kinetic data for 186 r;ztprhni 4-rq C bL,aiecn n--, n,,innnp their reactions, e.g., termination rate constants for osemiquinones.

Autoxidation/Chemical Oxidation
Free-radical chain reactions have been proposed during autoxidation of catechol(amines)s (82). From the pH dependence of oxygen consumption, the initiating step of an autoxidation reaction appears to involve electron transfer from the mono-anion of the catechol(amine) to molecular oxygen, e.g., Although several metal ions (e.g., Al3", Y3+, Cd2+, Ca2+, Mg2+, and Zn2+) have been employed to stabilize o-semiquinones in aqueous (86) and nonaqueous media (87), we feel that either Mg2+ or Zn2+ is more likely to be useful in biological systems, and consequently only Zn2+and Mg2+-complexed o-semiquinones in aqueous media are discussed in this review.
Except at high pH, o-semiquinone radicals are transient, decaying rapidly via disproportionation to give the catechol and o-quinone. I::c/r + 02 A more recent study, however, suggests that this electron transfer to oxygen is metal-catalyzed (83). The superoxide radical formed in the initiating reaction also is scavenged by catechol(amine) (84). HO .0 R v~~+ 02 / H202 HO (3) Radicals can be generated by either static or flow measurements (85). Static oxidation involves addition of the catechol(amine) or catechol estrogen to an oxygen-saturated solution of sodium hydroxide (76,78,79). Semiquinones are observed fairly easily at high pH because of the slow rate of dismutation of radical anions. Autoxidation also allows the monitoring of secondary radical formation, i.e., semiquinones from 6-hydroxysubstituted catechol(amine). Alkaline hydrogen peroxide (a source of superoxide anion) is another effective way of oxidizing catecholamines and, possibly, catechol estrogens (84). Both sodium periodate and silver oxide are often used to oxidize catechols. Periodate is a twoelectron oxidizing agent, but silver oxide appears to act as a one-electron oxidant (75,76).
Enzymatic Oxidation o-Semiquinones formed during enzymatic oxidation have previously been detected by ESR using continuous flow methods (85). This procedure entails the use of large volumes of substrate and enzyme. Recently, we have developed a spin-stabilization procedure by which o-semiquinones (formed enzymatically) can be stabilized by chelation through the use of diamagnetic dior trivalent ions (73).
HO / (5) For dopa, the measured radical half-life was 2 msec for a steady-state concentration of 2.5 ,uM, corresponding to a second-order termination rate constant of 2 x 0 M1 sec- (Fig. 4). However, Zn2+-complexed dopa semiquinone radicals are much less transient than the uncomplexed ones (88). Decay remains second-order, but the radical lifetime is now several seconds for a steady-state concentration of ca. 10-5 M. The calculated second-order rate constant is 1.1 x 104 M-1sec-1.  Chelation (or complexation) is therefore extremely effective in decreasing the rate of radical termination. The uncomplexed o-semiquinone at neutral pH has a rate constant over 10,000-fold greater. Thus, the complexed radical can be detected at rates of radical for- (4) mation 10,000 times lower than are necessary to detect I . I the uncomplexed o-semiquinone. This allows the use of static rather than flow systems (73). Whereas HRP/H202-dependent oxidation of catechol(amine) and catechol estrogens involves o-semiquinones as obligate intermediates (88), the tyrosinasecatalyzed oxidation proceeds via a two-electron oxidation (41) with the formation of o-semiquinones in a secondary reaction. HO  The enzyme activity in each system is not affected to any marked extent by the presence of Zn2+ or Mg2e ions (73).

Characterization and Identification of o-Semiquinones
Production of primary and secondary free radicals from the oxidation of catechols and the dopa and epinephrine classes of catecholamines is shown in Figure  5.
Ir Whereas the identification of semiquinones from catechols has been fairly straightforward (71,74), there existed several inconsistencies with regard to interpretation of spectra of o-semiquinones from dopa (89) and epinephrine (91). The major reasons for the observed inconsistencies were: the presence of more than one spectral species; the presence of magnetically inequivalent methylene protons in the amino acid side chain; and acid-base equilibria in the radicals.
For example, oxidation of dopa and its analogs can, depending on the conditions employed, give three major types of radical (D1-D3). The primary radical (D1) does not show the expected multiplicities or linewidths because of a combination of the magnetic inequivalence referred to and restricted rotation (46,90). This phenomenon is illustrated in Figure 6. At low temperature only one-half of the spectral lines are clearly visible, whereas when the temperature is increased the "missing lines" that were previously broadened are apparent and complete spectral analysis becomes possible. An additional complexity in the system is provided by the ionization of the amino group (pKa= 9) which causes a shift in the spectral parameters at high pH (Fig. 7).
Data for uncomplexed and complexed primary o-semiquinones of catechol(amine)s are given in Tables 1 and 2. The magnetic parameters ofthe complexes ( Table  2) are modified from those of the uncomplexed species, with the differences between them being fairly constant (74). For example, for the Zn2"-complexed species c43 and a' typically decrease by about 0.28 G in going to the complex while aP increases by about the same amount. ae also increases for Zn2+. Observation of satellite peaks from magnetic isotopes present in natural abundance [e.g., from 67Zn (4%,I = 5/2)] verified that complex formation is occurring. Also indicative of com- plex formation are the observed changes in g value. g Values are decreased in the complexes, consistent with spin density in a vacant metal orbital.
Although hyperfine couplings to aromatic protons, in particular a05, do not differ markedly for the majority of radicals studied, couplings to the methylene protons in a substituent at position 4 vary considerably, suggesting that the rotations of these protons can be quite restricted (Tables 1 and 2). For example, whereas the protons in a freely rotating alkyl substituent (such as the methyl group in 4-methyl o-benzosemiquinone) have a hyperfine splitting of about 4.8 G, the protons in the a-methyl dopa semiquinone radical [where R = CH2C(CH3)(NH3+)(CO2-)] have couplings as low as 2.2 G (the mean of splittings from two inequivalent protons). In general, the methylene proton couplings decrease as the bulk of the substituent on the carbon atom bearing the methylene protons increases. Secondary radicals C2 from catechols and D2 and D3 from dopa analogs have been detected during autoxidation (44,76) and enzyme oxidation (with tyrosinase); they are derived from hydroxycatechols, 6-hydroxydopa, and 5,6-dihydroxyindole respectively ( Table 3). The unusual spectrum of radical D2 again is a consequence of magnetic inequivalence and restricted rotation in a single radical (  solution gives transient, broad, poorly resolved spectra under most conditions (91). Use of ESR-spin stabilization, however, enabled us to obtain three types of radical (75). Their spectra and, for the most part, their structures, are distinct from the dopa series. The primary radical El lacks a second methylene hydrogen (Fig. 5) in the side chain, so that its spectrum is much narrower than that of Dl; spectra of the secondary radicals (E2 and E3) are, however, much greater because of a large hyperfine splitting from nitrogen following cyclization (note that the nitrogen coupling in D3 is much lower than in either E2 or E3 possibly due to additional spin-delocalization in the indole ring). Also notable is the failure to detect hydroxy-substituted semiquinones from the epinephrine class unlike the dopa or catechol series (Fig. 5). This is possibly due to a more rapid rate of cyclization of the precursor quinones. Magnetic parameters that characterize these secondary radicals are give in Table 5.
Adrenochrome is the stable 4-electron oxidation product of epinephrine. There is evidence for increased toxicity (e.g., myocardial necrosis) from adrenochrome in the presence of the reducing agents, ascorbic acid and cysteine (67). Ascorbic acid previously has been shown to reduce adrenochrome to leucoadrenochrome via a free radical mechanism. Adrenochrome forms both oneelectron reduced and one-electron oxidized radicals.
Oxidation radical The one-electron oxidized species has recently been identified during peroxidatic oxidation of adrenochrome (75). However, there is evidence for one-electron reduction of adrenochrome in microsomes containing NADPH (68). This proposed species apparently is oxidized by molecular oxygen forming superoxide and the parent compound (68). We have again chosen the spin stabilization approach in a chemical system (92) (Fig.   8). Magnetic parameters for the Zn2+and Mg2e-complexed species are in Table 6.
Semiquinone radicals have been detected for the first time from auto-and enzymatic oxidation of catechol estrogens (93); the spin stabilization approach was again crucial to detect semiquinones produced enzymatically.
Using either Zn2+ or Mg2e as complexing agents, we showed the production of semiquinones (VII (R = H), VIIa (R = H), VII (R = OH) and VIIa (R = OH)) during peroxidase/tyrosinase oxidation of catechol estrogens and estrogens (Figs. 9 and 10). The species VII (R = H) is characterized by three large hyperfine couplings to P-alicyclic protons ( at C-6 and C-9), whereas VII (R = OH) exhibits only two large couplings. The species VIIa (R = H) is characterized by only one large coupling to an alicyclic p-proton (C-9) and a significant coupling to an aromatic proton; species VIIa (R = OH) exhibits a similar pattern. From these spectra, one can unequivocally assign the coupling to specific alicyclic protons. The g-values are close to those reported for metal complexes of o-semiquinones from simple catechols ( Fig. 10) (71,74). Magnetic parameters that characterize each species are given in Table 7.

Reactions of o-Semiquinones
The spin stabilization approach has enabled us to study radical reactions of o-semiquinones in two enzymatic systems (HRP/H202 and tyrosinase/02). o-Semiquinone radicals were detected in high steady-state con- In systems containing catecholamine, HRP/H202, and phenol in moderate concentrations, the rate of semiquinone production showed a marked increase as a function of phenol concentration. This is consistent with the production of phenoxyl PhO-radieals which subsequently oxidizes the catecholamine: -e PhOH -) PhO-PhO-+ QH2 -PhOH + Q-+ 2H+ (10) (11) From photolytic systems, the rate constant kd was calculated as ca. 104 M-1 sec 1 (for complexed semiquinones) and as 108 M-1 sec-1 for uncomplexed semiquinone radicals.
During peroxidatic oxidation, with H202 limiting and constant substrate concentrations, the duration of the steady-state (t..) is linearly dependent on the initial concentration of hydrogen peroxide, [H202]. This allowed an estimate (Fig. 11)  In contrast, ascorbate (Fig. 12) and glutathione resulted in a lag time for semiquinone detection that was proportional to the concentration of reductant added. During the lag time, only radicals from the reducing agent are detected. Reaction between o-semiquinone and ascorbate was reported to be too slow to measure by pulse methods, although there is ESR evidence for the reduction ofascorbate by a semiquinone radical from 6-hydroxydopa (94).  'In this structure, the other ring hydrogen also is replaced by deuterium.   Mg2+-complexed semiquinones from the hydroxylation/oxidation of t-estradiol by tyrosinase/02; (D) Mg2+-complexed semiquinones from the hydroxylation/oxidation of 6a-hydroxyestradiol by tyrosinase/02. In spectrum C, radicals from 2-and 4-hydroxyestradiol are denoted by (x) and (0), respectively.
We verified in photolysis experiments that reactions of phenoxyl radicals with catechols, and of semiquinones with ascorbate, indeed occur. Formation of semiquinone is promoted by the presence of phenols, showing the ability of phenoxyl radical to oxidize catechols (Fig. 13). In contrast, semiquinone concentrations are strongly quenched by ascorbate and thiols. Radical decay is pseudo-first-order, indicating a direct reaction between the semiquinone and the reducing agent.
Although reactions between these primary o-semiquinones and oxygen have been postulated, we did not observe an increased oxygen consumption above background levels from autoxidation during peroxidatic oxidation of catechols. Since the o-semiquinone is an obligate intermediate in the peroxidase system, significant electron transfer to oxygen can therefore be ruled out.   Abscissa: reaction time; ordinate: ESR signal amplitude (proportional to free radical concentration). The time at which enzyme was added is indicated by the arrows. Individual measurements of At were reproducible to ± 10%. Conditions: 6 mM norepijnephrine, 28 nM horseradish peroxidase, 0.28 mM H202, 227 mM Zn2+ in acetic acid-acetate buffer, pH 5.0. The lag in semiquinone detection that is observed is proportional to the initial concentration of ascorbate (d).
Since semiquinones also can catalyze the direct reduction of H202, the feasibility of reduction of H202 by o-benzosemiquinone and p-semiquinone (e.g., daunorubicin semiquinone) was compared (95). We were able to demonstrate this reaction for daunorubicin semiquinone, but not for o-benzosemiquinone. fore, appear to reduce H202, o-semiquinones from hydroxy or amino substituted catechols may behave differently (by acquiring p-semiquinone character through resonance).
Quinones are electrophilic in nature (Fig. 2). They undergo addition reactions with the sulfhydryl groups present in DNA polymerase and also cause the inactivation of the enzyme (18,58). Indeed the selective toxicity of melanin precursors against melanoma cells has been shown to be due to formation of o-quinones (18). Production of pheomelanin involves the addition of cysteine to o-dopaquinone followed by subsequent polymerization (64). Catecholamine o-quinones have been shown to be more toxic to melanocytes than are aminochromes (probably due to lack of electrophilic reactive sites in the latter) (18). Reactions between o-quinones and amino acids (leading to "melanoproteins") form the basis of "browning reactions" that occur in fruits and vegetables (109).