Abstract
The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.
Highlights
A large number of sites worldwide are contaminated with high explosives as a result of large scale manufacturing and handling of these compounds
We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN
The spectral changes accompanying reduction of PETN reductase contrast with those seen for the photoreduction of Old Yellow Enzyme (OYE) in which the anionic red semiquinone is populated [24] but are similar to comparable titrations performed with bacterial morphinone reductase [25]
Summary
Chemicals and Enzymes—Complex bacteriological media were from Unipath, and all media were prepared as described by Sambrook et al [17]. For studies of the oxidative half-reaction, PETN reductase was titrated with sodium dithionite to the two-electron level and mixed with 2-cyclohexenone. Observed rate constants for flavin absorption changes accompanying (i) mixing of oxidized PETN reductase with NADPH or (ii) oxidation of reduced PETN reductase by oxidizing substrates were obtained from fits of the data to a single exponential expression. Reductive transients at 464 nm are strictly biphasic (see “Results”), but the fast first phase (charge transfer formation) contributes only a very small absorption change, making analysis using a biphasic expression inappropriate For this reason fitting using a single exponential expression was used, and analysis was performed on the kinetic transient in which the signal for the first 20 ms after the mixing event was truncated. Spectral changes resulting from the addition of ligand to PETN reductase indicated a 1:1
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