Mechanism of p-nitrosophenol reduction catalyzed by horse liver and human pi-alcohol dehydrogenase (ADH). Human pi-ADH as a quinone reductase.

The mechanism of reduction of p-nitrosophenol (pNSP) catalyzed by horse liver alcohol dehydrogenase (HADH) and human pi-alcohol dehydrogenase (pi-ADH) has been compared in transient and steady-state experiments. Our results indicate that pNSP reduction catalyzed by these two ADH proceeds by different mechanisms. In one mechanism, shown by Equation 1, pNSP is reduced to p-aminophenol (pAP) via two enzymatic steps (Steps 1 and 3), which are mediated by the nonenzymatic dehydration of p-N-hydroxyaminophenol (pN-OHAP) to 1,4-benzoquinoneimine (BQI) (Step 2). [formula: see text] Pathway (I) is proposed mainly for pi-ADH but can be catalyzed by HADH. However, Step 3 is catalyzed approximately 2 orders of magnitude more slowly by HADH than by pi-ADH. This conclusion is confirmed by the results, which indicate that pi-ADH very efficiently catalyzes the reduction of BQI and 1,4-benzoquinone (BQ) to the corresponding hydroquinones. The kinetic constants determined at pH 7.4 suggest that pi-ADH is a more efficient quinone reductase and nitroso reductase than it is an ethanol oxidase or acetaldehyde reductase. An alternative mechanism of pNSP reduction, shown by Equation 2, is suggested for HADH. In this mechanism, formation of the p-hydroxybenzylnitrenium ion (pNH+P) occurs at the active-site zinc ion of the enzyme (Step 2) and accelerates further nonenzymatic reduction to pAP or hydrolysis to BQ (Step 3). [formula: see text]

Alcohol dehydrogenase-dependent reduction of 2-nitrosoflurene and rearrangement of N-hydroxy-2-aminofluorene.

Various C-nitroso compounds are intermediates of arylamine oxidation or nitroarene reduction. Reductive metabolism of C-nitroso compounds to their corresponding hydroxylamines is a necessary step in the activation of these compounds to mutagenic end points. In this study, 2-nitrosofluorene (2-NOF) has been investigated as an aldehyde substrate analogue for horse liver alcohol dehydrogenase (HLADH). The reaction products are assigned on the basis of their UV/visible spectra and coelution with authentic standards in reversed-phase HPLC. The direct product of 2-NOF reduction is N-hydroxy-2-aminofluorene (N-OH-2-AF), which undergoes further reduction to 2-aminofluorene (2-AF) and rearrangement to 1- and 3-hydroxy-2-aminofluorene (1- and 3-OH-2-AF). The formation of these products is potently inhibited by pyrazole indicating the involvement of active-site zinc ion in the role of a Lewis acid catalyst. It is suggested that the rearrangement reaction occurs via an inner-sphere N-OH 2AF-Zn2+...E-coenzyme complex following the elimination of the hydroxyl group from the N-OH-2-AF intermediate and the hydrolysis of the fluorenyl nitrenium-derived carbocations to yield the hydroxy 2-AF products. Herein ADH is identified as a C-nitroso-reducing enzyme which must be considered in the mutagenic sequelae of nitro and nitrosoarenes.

The hydroxylation of tryptophan.

Products of the chemical hydroxylation of tryptophan by Fenton and Udenfriend reactions are similar to those obtained by ionizing radiation. When tryptophan is exposed to either of these systems, a mixture of four hydroxytryptophans, oxindole-3-alanine, and N-formylkynurenine is formed. This observation indicates that the hydroxyl radical attacks the aromatic nucleus as well as the 2 and 3 positions of the pyrrole ring. During gamma-radiolysis of nitrous oxide-saturated tryptophan solution and in the absence of oxygen or ferric edta, the hydroxyl radical adduct (or hydroxycyclohexadienyl radical) of tryptophan undergoes dimerization and polymerization, which results in a yellow product with maximal absorbance at 425 nm. In the presence of ferric edta, or in a Fenton system, the hydroxyl radical adduct disproportionates, and hydroxylated derivatives are formed. The yields of the hydroxytryptophans are proportional to the concentration of ferric edta to a limiting yield of 54% of the theoretical yield, which is taken to be one hydroxylated product per two hydroxyl radicals. Under these conditions, 4-, 5-, 6-, and 7-hydroxy-derivatives of tryptophan are found in the proportion 4:2:2:3, respectively. The presence of dioxygen during gamma-radiolysis increases the yield of N-formylkynurenine, but does not affect the total yield of hydroxytryptophans. Similarly, tryptophan subjected to the Udenfriend reaction yields 4-, 5-, 6-, and 7-hydroxytryptophan and N-formylkynurenine in approximately equal amounts.

The hydroxylation of phenylalanine and tyrosine: a comparison with salicylate and tryptophan.

The hydroxylation of phenylalanine by the Fenton reaction and gamma-radiolysis yields 2-hydroxy-, 3-hydroxy-, and 4-hydroxyphenylalanine (tyrosine), while the hydroxylation of tyrosine results in 2,3- and 3,4-dihydroxyphenylalanine (dopa). Yields are determined as a function of pH and the presence or absence of oxidants. During gamma-radiolysis and the Fenton reaction the same hydroxylated products are formed. The final product distribution depends on the rate of the oxidation of the hydroxyl radical adducts (hydroxycyclohexadiene radicals) relative to the competing dimerization reactions. The pH profiles for the hydroxylations of phenylalanine and tyrosine show a maximum at pH 5.5 and a minimum around pH 8. The lack of hydroxylated products around near pH 8 is due to the rapid oxidation of dopa to melanin. The relative abilities of iron chelates (HLFe(II) and HLFe(III) to promote hydroxyl radical formation from hydrogen peroxide are nitrilotriacetate (nta) greater than ethylenediaminediacetate (edda) much greater than hydroxyethylethylenediaminetriacetate greater than citrate greater than ethylenediaminetetraacetate greater than diethylenetriaminepentaacetate greater than adenosine 5'-triphosphate greater than pyrophosphate greater than adenosine 5'-diphosphate greater than adenosine 5'-monophosphate. The high activity of iron-nta and -edda chelates is explained by postulating the formation of a ternary Fe(III)-L-dopa complex in which dopa reduces Fe(III). The hydroxylations of phenylalanine and tyrosine are similar to that of salicylate (Z. Maskos, J. D. Rush, and W. H. Koppenol, 1990, Free Radical Biol. Med. 8, 153-162) and tryptophan (preceding paper) in that oxidants augment the formation of hydroxylated products by catalyzing the dismutation of hydroxyl radical adducts to the parent compound and a stable hydroxylated product. A comparison of salicylate and the amino acids tryptophan, phenylalanine, and tyrosine clearly shows that salicylate is the best indicator of hydroxyl radical production.

The superoxide dismutase activities of two higher valent manganese complexes, MnIV desferrioxamine and MnIII-cyclam.

A green manganese desferrioxamine complex is rapidly formed at room temperature upon stirring freshly precipitated manganese dioxide in a solution of the ligand. Spectral studies and low-temperature ESR indicate that this compound, which has been previously described as a manganese(III) complex, is better characterized as containing tetravalent manganese. The complex appears to form oligomers in solution. The extinction coefficient at 635 nm is 137 +/- 6 M-1 cm-1 (per manganese) at pH 7.8 and 88 +/- 4 M-1 s-1 at pH 6.6 after purification by chromatography. The superoxide dismutase activity was measured and compared to that of mononuclear manganese(III) 1,4,8,11-tetraazacyclodecane (cyclam). The catalytic rate constants for superoxide dismutase activity are 1.7 x 10(6) M-1 s-1 and 2.9 x 10(6) M-1 s-1 for the desferrioxamine and the cyclam complexes, respectively.

The hydroxylation of the salicylate anion by a Fenton reaction and T-radiolysis: a consideration of the respective mechanisms.

The yield of 2,3- and 2,5-dihydroxybenzoates (dHB's) from the reaction of .OH radicals with salicylate (SA) ions has been measured as a function of pH and in the presence of oxidants. Under steady-state radiolysis conditions, the production of these products occurs via the reactions .OH + SA----HO-SA. (radical adduct) HO-SA. H+.OH+----2-carboxyphenoxyl radical (SA.) + H2O HO-SA. + SA.----2,3-/2,5-dHB + SA The addition of the oxidants O2, Fe3+ edta, or Fe(CN)63- increases the relative yield of 2,5-dHB/2,3-dHB from about 0.2 to 1. A model to account for this effect is presented. Steady-state radiolyses of 3- and 4-hydroxybenzoate give dihydroxybenzoate products consistent with the phenol group being an ortho-para director in the electrophilic attack of the hydroxyl radical on the aromatic ring. A comparison of product distributions from the reaction of ferrous edta with hydrogen peroxide using salicylate as a scavenger strongly suggests that the same hydroxyl radical adducts are formed as in the radiation experiments.