Žilvinas Anusevičius

Reduction of aliphatic nitroesters and N-nitramines by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships.

Enterobacter cloacae PB2 NADPH:pentaerythritol tetranitrate reductase (PETNR) performs the biodegradation of explosive organic nitrate esters via their reductive denitration. In order to understand the enzyme substrate specificity, we have examined the reactions of PETNR with organic nitrates (n = 15) and their nitrogen analogues, N‐nitramines (n = 4). The reactions of these compounds with PETNR were accompanied by the release of 1–2 mol of nitrite per mole of compound, but were not accompanied by their redox cycling and superoxide formation. The reduction rate constants (kcat/Km) of inositol hexanitrate, diglycerol tetranitrate, erythritol tetranitrate, mannitol hexanitrate and xylitol pentanitrate were similar to those of the established PETNR substrates, PETN and glycerol trinitrate, whereas the reactivities of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine and octahydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine were three orders of magnitude lower. The log kcat/Km value of the compounds increased with a decrease in the enthalpy of formation of the hydride adducts [ΔHf(R–O–N(OH)O−) or ΔHf(R1,R2 > N–N(OH)O−)], and with an increase in their lipophilicity (octanol/water partition coefficient, log Pow), and did not depend on their van der Waals’ volumes. Hydrophobic organic nitroesters and hydrophilic N‐nitramines compete for the same binding site in the reduced enzyme form. The role of the hydrophobic interaction of PETNR with glycerol trinitrate was supported by the positive dependence of glycerol trinitrate reactivity on the solution ionic strength. The discrimination of nitroesters and N‐nitramines according to their log Pow values seems to be a specific feature of the Old Yellow Enzyme family of flavoenzymes.

DT-diaphorase catalyzes N-denitration and redox cycling of tetryl

Rat liver DT‐diaphorase (EC 1.6.99.2) catalyzed reductive N‐denitration of tetryl (2,4,6‐tri‐nitrophenyl‐N‐methylnitramine) and 2,4‐dinitrophenyl‐N‐methylnitramine, oxidizing the excess of NADPH. The reactions were accompanied by oxygen consumption and superoxide dismutase‐sensitive reduction of added cytochrome c and reductive release of Fe2+ from ferritin. Quantitatively, the reactions of DT‐diaphorase proceeded like single‐electron reductive N‐denitration of tetryl by ferredoxin:NADP+ reductase (EC 1.18.1.2) (Shah, M.M. and Spain, J.C. (1996) Biochem. Biophys. Res. Commun. 220, 563–568), which was additionally checked up in this work. Thus, although reductive N‐denitration of nitrophenyl‐N‐nitramines is a net two‐electron (hydride) transfer process, DT‐diaphorase catalyzed the reaction in a single‐electron way. These data point out the possibility of single‐electron transfer steps during obligatory two‐electron (hydride) reduction of quinones and nitroaromatics by DT‐diaphorase.

Interaction of quinones with Arabidopsis thaliana thioredoxin reductase

In view of the ubiquitous role of the thioredoxin/thioredoxin reductase (TRX/TR) system in living cells, the interaction of Arabidopsis thaliana NADPH-thioredoxin reductase (EC 1.6.4.5) with quinones, an important class of redox cycling and alkylating xenobiotics, was studied. The steady-state reactions of A. thaliana TR with thioredoxin (TRX) and reaction product NADP+ inhibition patterns were in agreement with a proposed model of E. coli enzyme (B.W. Lennon, C.H. Williams, Jr., Biochemistry, vol. 35 (1996), pp. 4704-4712), that involved enzyme cycling between four- and two-electron reduced forms with FAD being reduced. Quinone reduction by TR proceeded via a mixed single- and two-electron transfer, the percentage of single-electron flux being equal to 12-16%. Bimolecular rate constants of quinone reduction (kcat/km) and reaction catalytic constants (kcat) increased upon an increase in quinone single-electron reduction potential. E(1)7. In several cases, the kcat of quinone reduction exceeded kcat of TRX reduction, suggesting that quinones intercepted electron flux from TR to TRX. Incubation of reduced TR with alkylating quinones resulted in a rapid loss of TRX-reductase activity, while quinone reduction rate was unchanged. In TRX-reductase and quinone reductase reactions of TR, NADP+ exhibited different inhibition patterns. These data point out that FAD and not the catalytic disulfide of TR is responsible for quinone reduction, and that quinones may oxidize FADH2 before it reduces catalytic disulfide. Most probably, quinones may oxidize the two-electron reduced form of TR, and the enzyme may cycle between two-electron reduced and oxidized forms in this reaction. The relatively high rate of quinone reduction by A. thaliana thioredoxin reductase accompanied by their redox cycling, confers pro-oxidant properties to this antioxidant enzyme. These factors make plant TR an attractive target for redox active and alkylating pesticide action.

The Study of NADPH-Dependent Flavoenzyme-Catalyzed Reduction of Benzo(1,2-c)1,2,5-oxadiazole N-Oxides (Benzofuroxans)

The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans; BFXs) towards mammalian single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and two-electron (hydride) transferring NAD(P)H:quinone oxidoreductase (NQO1) was examined in this work. Since the =N+ (→O)O− moiety of furoxan fragments of BFXs bears some similarity to the aromatic nitro-group, the reactivity of BFXs was compared to that of nitro-aromatic compounds (NACs) whose reduction mechanisms by these and other related flavoenzymes have been extensively investigated. The reduction of BFXs by both P-450R and NQO1 was accompanied by O2 uptake, which was much lower than the NADPH oxidation rate; except for annelated BFXs, whose reduction was followed by the production of peroxide. In order to analyze the possible quantitative structure-activity relationships (QSARs) of the enzymatic reactivity of the compounds, their electron-accepting potency and other reactivity indices were assessed by quantum mechanical methods. In P-450R-catalyzed reactions, both BFXs and NACs showed the same reactivity dependence on their electron-accepting potency which might be consistent with an “outer sphere” electron transfer mechanism. In NQO1-catalyzed two-electron (hydride) transferring reactions, BFXs acted as more efficient substrates than NACs, and the reduction efficacy of BFXs by NQO1 was in general higher than by single-electron transferring P-450R. In NQO1-catalyzed reactions, QSARs obtained showed that the reduction efficacy of BFXs, as well as that of NACs, was determined by their electron-accepting potency and could be influenced by their binding mode in the active center of NQO1 and by their global softness as their electronic characteristic. The reductive conversion of benzofuroxan by both flavoenzymes yielded the same reduction product of benzofuroxan, 2,3-diaminophenazine, with the formation of o-benzoquinone dioxime as a putative primary reductive intermediate, which undergoes a further reduction process. Overall, the data obtained show that by contrast to NACs, the flavoenzyme-catalyzed reduction of BFXs is unlikely to initiate their redox-cycling, which may argue for a minor role of the redox-cycling-type action in the cytotoxicity of BFXs.

Quinones and nitroaromatic compounds as subversive substrates of Staphylococcus aureus flavohemoglobin

Abstract In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe3+, metHb) convert NO. into nitrate at the expense of NADH and O2. FHbs contribute to bacterial resistance to nitrosative stress. Therefore, inhibition of FHbs functions may decrease the pathogen virulence. We report here a kinetic study of the reduction of quinones and nitroaromatic compounds by S. aureus FHb. We show that this enzyme rapidly reduces quinones and nitroaromatic compounds in a mixed single‐ and two‐electron pathway. The reactivity of nitroaromatics increased upon an increase in their single‐electron reduction potential (E17), whereas the reactivity of quinones poorly depended on their E17 with a strong preference for a 2‐hydroxy‐1,4‐naphthoquinone structure. The reaction followed a ‘ping‐pong’ mechanism. In general, the maximal reaction rates were found lower than the maximal presteady‐state rate of FAD reduction by NADH and/or of oxyhemoglobin (HbFe2+O2) formation (˜130 s−1, pH 7.0, 25 °C), indicating that the enzyme turnover is limited by the oxidative half‐reaction. The turnover studies showed that quinones prefreqently accept electrons from reduced FAD, and not from HbFe2+O2. These results suggest that quinones and nitroaromatics act as ‘subversive substrates’ for FHb, and may enhance the cytotoxicity of NO. by formation of superoxide and by diverting the electron flux coming from reduced FAD. Because quinone reduction rate was increased by FHb inhibitors such as econazole, ketoconazole, and miconazole, their combined use may represent a novel chemotherapeutical approach. Graphical abstract Quinones as subversive substrates for flavohemoglobin. Figure. No Caption available. HighlightsS. aureus flavohemoglobin reduces quinones and nitroaromatics in 1e‐/2e‐ way.Quinones accept electrons from reduced FAD, and not from oxyhemoglobin moiety.Quinone reduction rate was increased by flavohemoglobin azole inhibitors.The combination of azoles and quinones may represent a novel chemotherapy approach.