Richard J. Harris

The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme.

The reaction of heterotetrameric sarcosine oxidase (TSOX) of Arthrobactor sp. 1-IN has been studied by stopped-flow spectroscopy, with particular emphasis on the reduction of the enzyme by sarcosine. Expression of the cloned gene encoding TSOX in Escherichia coli enables the production of TSOX on a scale suitable for stopped-flow studies. Treatment of the enzyme with sulfite provides the means for selective formation of a flavin-sulfite adduct with the covalent 8alpha-(N(3)-histidyl)-FMN. Formation of the sulfite-flavin adduct suppresses internal electron transfer between the noncovalent FAD (site of sarcosine oxidation) and the covalent FMN (site of enzyme oxidation) and thus enables detailed characterization of the kinetics of FAD reduction by sarcosine using stopped-flow methods. The rate of FAD reduction displays a simple hyperbolic dependence on sarcosine concentration. Studies in the pH range 6.5-10 indicate there are no kinetically influential ionizations in the enzyme-substrate complex. A plot of the limiting rate of flavin reduction/the enzyme-substrate dissociation constant (k(lim)/K(d)) versus pH is bell-shaped and characterized by two macroscopic pK(a) values of 7.4 +/- 0.1 and 10.4 +/- 0.2: potential candidates for the two ionizable groups are discussed with reference to the structure of monomeric sarcosine oxidase (MSOX). The kinetic data are discussed with reference to potential mechanisms for the oxidation of amine molecules by flavoenzymes. Additionally, kinetic isotope effect studies of the rate of C-H bond breakage suggest that a ground-state quantum tunneling mechanism for H-transfer, facilitated by the low-frequency thermal motions of the protein molecule, accounts for C-H bond cleavage by TSOX. TSOX thus provides another example of C-H bond breakage by ground-state quantum tunneling, driven by protein dynamics [vibrationally enhanced ground-state quantum tunneling (VEGST)], for the oxidation of amines by enzymes.

Summary We determined the association of uranium with bacteria isolated from the Waste Isolation Pilot Plant (WIPP), Carlsbad, New Mexico, and compared this with known strains of halophilic and non-halophilic bacteria and archaea. Examination of the cultures by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS) showed uranium accumulation extracellularly and/or intracellularly to a varying degree. In Pseudomonas fluorescens and Bacillus subtilis uranium was associated with the cell surface and in the latter it was present as irregularly shaped grains. In Halobacterium halobium , the only archeon studied here, uranium was present as dense deposits and with Haloanaerobium praevalens as spikey deposits. Halomonas sp . isolated from the WIPP site accumulated uranium both extracellularly on the cell surface and intracellularly as electron-dense discrete granules. Extended X-ray absorption fine structure (EXAFS) analysis of uranium with the halophilic and non-halophilic bacteria and archaea showed that the uranium present in whole cells was bonded to an average of 2.4±0.7 phosphoryl groups at a distance of 3.65±0.03 Å. Comparison of whole cells of Halomonas sp . with the cell wall fragments of lysed cells showed the presence of a uranium bidentate complex at 2.91±0.03 Å with the carboxylate group on the cell wall, and uranyl hydroxide with U-U interaction at 3.71±0.03 Å due to adsorption or precipitation reactions; no U-P interaction was observed. Addition of uranium to the cell lysate of Halomonas sp . resulted in the precipitation of uranium due to the inorganic phosphate produced by the cells. These results show that the phosphates released from bacteria bind a significant amount of uranium. However, the bacterially immobilized uranium was readily solubilized by bicarbonate with concurrent release of phosphate into solution.

Research on venomous animals has mainly focused on the molecular, biochemical, and pharmacological aspects of venom toxins. However, it is the relatively neglected broader study of evolutionary ecology that is crucial for understanding the biological relevance of venom systems. As fish have convergently evolved venom systems multiple times, it makes them ideal organisms to investigate the evolutionary ecology of venom on a broader scale. This review outlines what is known about how fish venom systems evolved as a result of natural enemy interactions and about the ecological consequences of evolving a venom system. This review will show how research on the evolutionary ecology of venom in fish can aid in understanding the evolutionary ecology of animal venoms more generally. Further, understanding these broad ecological questions can shed more light on the other areas of toxinology, with applications across multiple disciplinary fields.

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.