Nordihydroguaiaretic acid (NDGA), one of the most efficient inhibitors of lipoxygenases, is shown, by electron paramagnetic resonance, circular dichroism, and fluorescence studies, to reduce the catalytically active ferric soybean lipoxygenase 1 (Eox) to the inactive ferrous form (Ered). In decreasing order of reactivity, the following also reduce Eox: catechol greater than hydroquinone greater than 2,6-di-tert-butyl-4-methylphenol greater than esculetin greater than caffeic acid approximately equal to alpha-tocopherol greater than norepinephrine greater than dithiothreitol. The reduction of Eox by NDGA (kappa = 8.1 X 10(6) M-1 S-1, pH 9.0, 25 degrees C) is almost as fast as the Eox-catalyzed conversion of linoleate (LH) to 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate (LOOH) and the oxidation of Ered by LOOH to give Eox. Thus, NDGA can efficiently inhibit the Eox-catalyzed conversion of LH to LOOH by reducing Eox to the inactive Ered, thereby diminishing the turnover rate. Lipoxygenase catalyzes the oxidation of NDGA by LOOH at a rate that is consistent with the independently determined rate constant for the reduction of Eox by NDGA. All four reducing equivalents from the two catechol groups in NDGA can be utilized in the reduction of Eox, leading to the consumption of 4 mol of LOOH/mol of NDGA initially present. Because the catalytically inactive Ered is oxidized by fatty acid hydroperoxides (e.g., LOOH) to give the active Eox, reducing agents such as NDGA are most effective as lipoxygenase inhibitors at low hydroperoxide concentrations. Our results suggest that in vivo, where lipid hydroperoxides are maintained at low steady-state levels, reduction of lipoxygenase from the ferric to ferrous state may be important in the regulation of lipoxygenase activity and hence leukotriene biosynthesis.

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The solution chemistry of AX5)alkyl flavinium cations and radical species formed by their le-reduction are discussed. Previously unknown, the 4a-flavine hydroperoxides are established to be formed on reaction of M5) alkyl flavinium cations with H202 or on reaction of M5) alkyl-1,5-dihydroflavines with 3°2. The stability of the 4a-flavine hydroperoxide species is exemplified in the isolation and characterization of 4a-hydroperoxy-1%5)ethyl-3-methyllumiflavine. 4a-Flavine hydroperoxide compounds are shown to be stronger oxidants than H202, and to undergo a chemiluminescent reaction in the presence of an aldehyde. Preliminary observations on the chemiluminescent reaction of 4a-flavine hydroperoxides + RCHO are provided, and these are compared to those in the literature dealing with the bioluminescence of bacterial luciferase in the presence of 3O2 and RCHO.Peroxy adducts of oxidized flavine have been proposed (1) as intermediates in the 302 oxidation of 1,5-dihydroflavines (2), the mechanism of external flavomonooxygenases (3, 4), and the mechanism of bacterial luciferase (5). Proposals of mechanisms (1, 2, 3, 5) have dealt most often with the 4a-hydroperoxy flavine. Studies directed toward the chemistry of peroxy flavine compounds have not been forthcoming for the simple reason that none were known. We report herein the simple synthesis of 4a-hydroperoxy adducts of 5-ethyl-3-methyllumiflavine (FlC2H5-OOH) and analogs (FIR-OOH) and report evidence for their involvement as intermediates in the 3°2 oxidation of N(5)-alkyl-1,5-dihydrolumiflavine (6). Also of considerable interest is our finding of light emission on reaction of FlR-OOH compounds with aldehydes (models for bacterial luciferase).It has been appreciated for some time that N(5)-alkyl flavinium cations exist in solution in equilibrium with their pseudo-base forms (7). For N(5)-CD3 lumiflavine (Fl0xCD3), the equilibrium of Eq. 1 (X = H) has been established (8). It occurred to us that FIR-OOH could be prepared simply by the addition of H202 to an F10oxR species (Eq. 1, X = OH). Since FIR-OOH would be in equilibrium with Fl ¶1,,R, a knowledge of the chemistry of the latter was desired. In brief, our findings (8) [3]

The interaction of the sulfone of penicillanic acid with the TEM-2 beta-lactamase from Escherichia coli has been investigated as a function of pH between pH 7.0 and 9.6. The first-formed acyl-enzyme suffers one of three fates: deacylation, tautomerization to a bound enamine that transiently inhibited the enzyme, and a process (possibly transimination) that leads to enzyme inactivation. The observed changes in ultraviolet absorbance are consistent with the initially observed product of deacylation being the enamine tautomer (4) of the imine from malonsemialdehyde and penicillamine sulfinate. The same enamine can be generated nonenzymically from the sulfone at high pH. The transiently inhibited enzyme appears to be the same enamine attached to the enzyme by an ester linkage. The rather complex kinetic behavior can be deconvuluted by exploiting the effect of pH on the partitioning of the acyl-enzyme between deacylation and the transiently inhibited form of the enzyme. The pathways followed by penicillanic acid sulfone provide a model for the behavior of a number of other reagents that inactivate the beta-lactamase.

The synthesis of N5-methyl-and P5-ethyl-1,5-dihydroflavin mononucleotides is reported. These compounds show no bioluminescence activity with bacterial luciferase. This feature is interpreted in terms of steric hindrance between the M5-alkyl group and a hydrogen bonding group at the active site of -the luciferase. The chemiluminescence observed on reaction of N5-alkyl-1,5-dihydroflavins with oxygen and aldehydes has been shown to occur via formation of a mixed peroxide of flavin and aldehyde and to be associated with a primary deuterium isotope effet when [1-2Hlaldehyde is substituted for aldehyde. The time course for light emission has been compared for aldehyde and ketone substrates. The suggestion is entertained that the peroxide bond of 4a-hydroperoxyflavin is sufficiently polarized to allow this species to act as the oxidant per se at the active site of mixed function oxidases. The second-order rate constants for reaction of hydroperoxides with thioxane and I-are compared. 4a-Hydroperoxy-3,5-dimethyllumiflavin is shown to convert thioxane to its sulfoxide 1.8 X 105 times faster than t-butyl hydroperoxide. In a previous paper (1), we described the synthesis (Eq. 1) and characterization of 4a-hydroperoxy-5-ethyl-3-methyllumiflavin (4a-FlC2H5-OOH). This isolation and characterization of the flavin hydroperoxide provides irrefutable evidence that 4a-hydroperoxide may be formed on oxidation of a 1,5-dihydroflavin (1, 2).