Brenda Walker Griffin

Free radical intermediate in the N-demethylation of aminopyrine by horseradish peroxidase—hydrogen peroxide

The oxidative cleavage of alkyl groups on nitrogen, oxygen and sulfur to their corresponding aldehydes has been considered to be a characteristic mono-oxygenation reaction of liver microsomal cytochrome P-450, requiring NADPH and molecular oxygen [ I] . Kadlubar et al. [2] first reported that cumene hydroperoxide and various other organic hydroperoxides could replace both NADPH and O2 in certain cytochrome P-450-catalyzed N-demethylations. Other hydroperoxide-supported activities of liver microsomal cytochrome P-450, including O-dealkylation and aromatic hydrocarbon hydroxylase activities, analogous to the O2 /NADPH requiring reactions, have since been reported [3,4].. Since a peroxidase activity of liver microsomal cytochrome P-450 has been implicated by these findings, we have undertaken a study of the N-demethylase activity of other hemeproteins as possible models for the liver enzyme. The first report of aminopyrine N-demethylation by hydrogen peroxide catalyzed by horseradish peroxidase (HRP) was made by Gillette et al. [5] who did not further characterize this activity. Catalase was shown by Kadlubar et al. [2] to exhibit a significant aminopyrine iV-demethylase activity in the presence of those organic peroxides which also supported the cytochrome P-450-catalyzed reaction.

Chloroperoxidase-catalyzed halogenation of antipyrine, a drug substrate of liver microsomal cytochrome P-450☆

Abstract Chloroperoxidase catalyzes halogenation of antipyrine at the 4-position with stoichiometric H 2 O 2 , in the presence of excess KCl or KBr; the pH optima of these reactions, near pH 4.0, are characteristic of other halogenation activities of this enzyme. In the presence of KCl or KBr, antipyrine was an effective inhibitor of H 2 O 2 decomposition catalyzed by chloroperoxidase. Under similar experimental conditions, 4-halogenation of antipyrine also occurred in the absence of chloroperoxidase with stoichiometric NaOCl, or enzymatically with horseradish peroxidase in the presence of H 2 O 2 and excess KBr (but not KCl). As observed previously for chloroperoxidase, horseradish peroxidase catalyzed oxidation of Br − by H 2 O 2 to Br 2 , readily detected in the presence of excess Br − by the intense uv absorbance of Br 3 − . These nonenzymatic and enzymatic halogenating systems could also effect N-demethylation of antipyrine, with complete release of formal-dehyde requiring a severalfold molar excess of NaOCl or H 2 O 2 respectively. These results and data obtained with 4-bromoantipyrine indicated that the methyl group is cleaved subsequent to halogenation. Since the halide anion was absolutely required for the enzymatic N-demethylation reactions, it appears that the enzymatically generated halogenating species is also responsible for N-demethylation, which is much less favorable than halogenation of antipyrine. These results parallel qualitatively the relative extents of 4-hydroxylation and N-demethylation of antipyrine catalyzed by liver microsomal cytochrome P -450 in vivo and in vitro and provide evidence for similar functions of chloroperoxidase and cytochrome P -450 as catalysts of: (1) dehydrogenation reactions and (2) insertion of an electonegative atom (Cl or 0) into an organic compound.

Mechanism of halide-stimulated activity of chloroperoxidase evidence for enzymatic formation of free hypohalous acid.

In acidic solutions, bromide stimulates H2O2 decomposition catalyzed by chloroperoxidase or horseradish peroxidase, altogether similar to stimulation, by chloride and bromide, of H2O2 oxidation by HOCl. Low levels of an ultra-pure NaCl inhibited chloroperoxidase catalatic activity whereas very high concentrations of this salt stimulated the reaction. The stimulation reflects low bromide contamination of the ultra-pure NaCl, which is considerably less than Br levels in most AR grade chloride salts. In all of these systems, bromide functions as a catalyst which is first oxidized to Br2 by HOX (added directly or generated enzymatically), and subsequently regenerated by H2O2 reduction of Br2. These results provide strong support for a dissociable species, most likely HOX, as the first product of chloroperoxidase catalyzed oxidation of Br- and Cl- by H2O2.

Chlorination of NADH: similarities of the HOCl-supported and chloroperoxidase-catalyzed reactions

The chloroperoxidase-catalyzed reactions of NAD(P)H with H2O2 in the presence of Cl- or Br- have been characterized. With 1 mol H2O2 per mol of NADH, one atom of 36Cl was incorporated into the 264-nm-absorbing intermediate product. This species was oxidized enzymatically by a second mole of H2O2 to a species distinct from NAD+, which retained one Cl atom. Spectroscopically identical species were also produced by reaction of NADH with one and two molar ratios of HOCl, respectively. These data indicate that, with respect to halogenation activities, chloroperoxidase functions similarly to myeloperoxidase, i.e., produces HOCl as the first product of Cl- oxidation by H2O2. Moreover, rapid chlorination of NAD(P)H followed by oxidation may be an important and highly lethal microbicidal effect of HOCl produced by myeloperoxidase in activated neutrophils.

Evidence for a radical mechanism of halogenation of monochlorodimedone catalyzed by chloroperoxidase

A radical species of monochlorodimedone has been characterized by its high reactivity with molecular O2. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous H2O2 only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of H2O2 added and O2 consumed. However, with excess Br- and horseradish peroxidase, or high Br- or Cl- and chloroperoxidase, a 1:1 stoichiometry between H2O2 and substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per H2O2. Moreover, in the absence of halide, at substrate:H2O2 ratios greater than 2.0, chloroperoxidase catalyzed significant O2 uptake; this enzyme-dependent autoxidation of monochlorodimedone also occurred in the presence of Cl- or Br-, when H2O2 was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Commun. 116, 873-879], strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOCl formation catalyzed by chloroperoxidase are considered.

Evidence for a free radical mechanism of N-demethylation of N,N-dimethylaniline and an analog by hemeprotein-H2O2 systems☆

Abstract Horseradish peroxidase and metmyoglobin catalyze the H 2 O 2 -supported N -demethylation of N,N -dimethylaniline and N,N -dimethyl- p -toluidine. The catalytic activities of horseradish peroxidase are more than 100-fold larger than those of metmyoglobin or those previously reported for liver microsomal cytochrome P -450. Distinct free radical species of these N -methyl substrates were detected with both catalysts. These findings establish the general validity of a recently proposed free radical mechanism of oxidative N -demethylation ( Griffin, B. W., and Ting, P. L., Biochemistry (1978), 2206–2211 ), which is quite different from that previously suggested for the analogous cytochrome P -450-dependent reactions.

Spin trapping evidence for free radical oxidants of aminopyrine in the metmyoglobin-cumene hydroperoxide system.

Preliminary accounts of this work were presented at the American Chemical Society Southeast—Southwest Regional Meeting, October 29–31, 1975, Memphis, TN and at the American Chemical Society Southwest Regional Meeting, December 5–7, 1977, Little Rock, AR

Electron paramagnetic resonance study of the oxidation of N-methyl-substituted aromatic amines catalyzed by hemeproteins

Abstract The oxidation of N-mono- and dimethyl-substituted toluidines and aniline by H2O2, catalyzed by horseradish peroxidase or metmyoglobin, produces organic free radicals, detectable by electron paramagnetic resonance spectroscopy at room temperature. The radical cation of N,N-dimethyl-p-toluidine was conclusively identified, but the other resolved EPR signals were assigned to radical cations of radical dimerization products, e.g., N,N,N′,N′-tetramethylbenzidine formed from N,N-dimethylaniline. The N-demethylase activities of metmyoglobin were found to be uniformly smaller than those of horseradish peroxidase, consistent with the much faster reaction of the latter hemeprotein with H2O2. Detection of the monomeric radical cation of N,N-demethyl-p-toluidine correlated with the largest rate of N-demethylation among this class of compounds. These findings emphasize the importance of radical stability (provided, for example, by the para methyl substituent) on subsequent competing reactions of the radical cation of the N-methyl substrate, i.e., one-electron oxidation leading to formaldehyde release or radical dimerization, which becomes more probable for the less stable radical intermediates. Attempts were made to correlate these results with data obtained for the O 2 NADPH -supported oxidation of these same substrates by liver microsomal cytochrome P-450. However, pronounced differences in physical state and kinetic properties of this heterogeneous, membrane-associated microsomal hemeprotein and the soluble “model” hemeprotein systems precluded firm conclusions concerning a radical mechanism of N-demethylation monooxygenase activities of microsomal fractions.

Electron acceptor function of O2 in radical N-demethylation reactions catalyzed by hemeproteins

Abstract In aerobic solutions, O 2 consumption correlated well with N-demethylation of N,N-dimethyl- p -toluidine catalyzed by horseradish peroxidase, in the presence or absence of H 2 O 2 . In the absence of added H 2 O 2 , superoxide dismutase stimulated, and catalase inhibited, both reactions; in the presence of H 2 O 2 , argon inhibition of formaldehyde production increased with increasing concentration of horseradish peroxidase. These results provide evidence for competing reactions of the enzymatically-generated substrate radical: oxidation by O 2 increases formaldehyde production, while radical dimerization decreases the yield of this product. Implications of these findings for similar reactions catalyzed by microsomal cytochrome P-450 are suggested.

Electron paramagnetic resonance probes of the radical decomposition of cumene hydroperoxide initiated by metmyoglobin

Abstract Electron paramagnetic resonance studies have provided evidence for metmyoglobin initiation of the radical decomposition of cumene hydroperoxide, carried out in buffered aqueous solutions at ambient temperatures. The radicals formed oxidize aminopyrine to a free radical, readily detected at acidic pH, or react with the spin trap nitrosobenzene. The only species so trapped was the cumyl radical (optimal pH, 9.0), previously observed in a similar spin-trapping study of the chemical decomposition of cumene hydroperoxide in organic solvents. The earlier proposal that the cumyl radical arises from breakdown of an initially formed, unstable phenylcumyloxy nitroxide is consistent with the experimental findings of this study. Moreover, it was shown that the decomposition of cumene hydroperoxide initiated by ferrous ion or by other heme compounds occurs by the same mechanism. Thus, the very low peroxidatic activities of several hemeproteins with cumene hydroperoxide involve oxidizing free radicals, unlike H 2 O 2 -dependent oxidations catalyzed by true hemeprotein peroxidases, in which enzyme species are the functional oxidants.