Studies of reduction mechanisms of quinones and nitroaromatic compounds by flavoenzymes dehydrogenases-transhydrogenases

In this work two flavoenzymes dehydrogenases-transhydrogenases are investigated - E. coli nitroreductase A (NfsA) and T. maritima thioredoxin reductase (TmTR). Recently, nitroreductases have acquired more interest concerning problems with biodegaration of nitroaromatic compounds and gene-directed enzyme prodrug therapy. TmTR-catalyzed reactions are also relevant due to interest of application of hyperthermophylic organisms in the synthesis of fuel and industrial chemicals. It was determined that two-electron reduction of quinones and nitroaromatic compounds by NfsA follows “ping-pong” kinetics and the rate-limiting step is the oxidative half-reaction. The experimental data are most consistent with a single-step (H-) hydride transfer mechanism in the reduction of quinones by NfsA. The obtained data argue for the major role of direct reduction by NADPH in the reduction of intermediate nitroso compound. The determined E07 value of FMN cofactor of NfsA (-0.215 V) is close to that of E. cloacae nitroreductase (-0.190 V). TmTR catalyzes mixed 1e- and 2e- reduction of quinones and nitroaromatic compounds. Reaction follows „ping-pong“ kinetics with a rate-limiting oxidative half-reaction. Accumulation of FAD semiquinone (FADH●) during the TmTR-catalyzed reduction of quinones indicates that the oxidation of FADH● may be a rate-limiting step in this reaction. The redox potential of FAD of TmTR, -0.230 V, is close to redox potentials of other low molecular mass thioredoxin reductases

Chinonų ir nitroaromatinių junginių redukcijos flavininėmis dehidrogenazėmis-transhidrogenazėmis mechanizmų tyrimai

In this work two flavoenzymes dehydrogenases-transhydrogenases are investigated - E. coli nitroreductase A (NfsA) and T. maritima thioredoxin reductase (TmTR). Recently, nitroreductases have acquired more interest concerning problems with biodegaration of nitroaromatic compounds and gene-directed enzyme prodrug therapy. TmTR-catalyzed reactions are also relevant due to interest of application of hyperthermophylic organisms in the synthesis of fuel and industrial chemicals. It was determined that two-electron reduction of quinones and nitroaromatic compounds by NfsA follows “ping-pong” kinetics and the rate-limiting step is the oxidative half-reaction. The experimental data are most consistent with a single-step (H-) hydride transfer mechanism in the reduction of quinones by NfsA. The obtained data argue for the major role of direct reduction by NADPH in the reduction of intermediate nitroso compound. The determined E07 value of FMN cofactor of NfsA (-0.215 V) is close to that of E. cloacae nitroreductase (-0.190 V). TmTR catalyzes mixed 1e- and 2e- reduction of quinones and nitroaromatic compounds. Reaction follows „ping-pong“ kinetics with a rate-limiting oxidative half-reaction. Accumulation of FAD semiquinone (FADH●) during the TmTR-catalyzed reduction of quinones indicates that the oxidation of FADH● may be a rate-limiting step in this reaction. The redox potential of FAD of TmTR, -0.230 V, is close to redox potentials of other low molecular mass thioredoxin reductases

Quinone- and nitroreductase reactions of [i]Thermotoga maritima[/i] thioredoxin reductase

The Thermotoga maritima NADH:thioredoxin reductase (TmTR) contains FAD and a catalytic disulfide in the active center, and uses a relatively poorly studied physiological oxidant Grx-1-type glutaredoxin. In order to further assess the redox properties of TmTR, we used series of quinoidal and nitroaromatic oxidants with a wide range of single-electron reduction potentials (E-7(1), -0.49-0.09 V). We found that TmTR catalyzed the mixed single- and two-electron reduction of quinones and nitroaromatic compounds, which was much faster than the reduction of Grx-1. The reactivity of both groups of oxidants increased with an increase in their E-7(1), thus pointing to the absence of their structural specificity. The maximal rates of quinone reduction in the steady-state reactions were lower than the maximal rates of reduction of FAD by NADH, obtained in presteady-state experiments. The mixed-type reaction inhibition by NAD(+) was consistent with its competition for a NADH binding site in the oxidized enzyme form, and also with the reoxidation of the reduced enzyme form. The inhibition data yielded a value of the standard potential for TmTR of -0.31 +/- 0.03 V at pH 7.0, which may correspond to the FAD/FADH(2) redox couple. Overall, the mechanism of quinone- and nitroreductase reactions of T. maritima TR was similar to the previously described mechanism of Arabidopsis thaliana TR, and points to their prooxidant and possibly cytotoxic role

Quinone- and nitroreductase reactions of Thermotoga maritima thioredoxin reductase

The Thermotoga maritima NADH:thioredoxin reductase (TmTR) contains FAD and a catalytic disulfide in the active center, and uses a relatively poorly studied physiological oxidant Grx-1-type glutaredoxin. In order to further assess the redox properties of TmTR, we used series of quinoidal and nitroaromatic oxidants with a wide range of single-electron reduction potentials (E-7(1), -0.49-0.09 V). We found that TmTR catalyzed the mixed single- and two-electron reduction of quinones and nitroaromatic compounds, which was much faster than the reduction of Grx-1. The reactivity of both groups of oxidants increased with an increase in their E-7(1), thus pointing to the absence of their structural specificity. The maximal rates of quinone reduction in the steady-state reactions were lower than the maximal rates of reduction of FAD by NADH, obtained in presteady-state experiments. The mixed-type reaction inhibition by NAD(+) was consistent with its competition for a NADH binding site in the oxidized enzyme form, and also with the reoxidation of the reduced enzyme form. The inhibition data yielded a value of the standard potential for TmTR of -0.31 +/- 0.03 V at pH 7.0, which may correspond to the FAD/FADH(2) redox couple. Overall, the mechanism of quinone- and nitroreductase reactions of T. maritima TR was similar to the previously described mechanism of Arabidopsis thaliana TR, and points to their prooxidant and possibly cytotoxic role

The Catalysis Mechanism of <i>E. coli</i> Nitroreductase A, a Candidate for Gene-Directed Prodrug Therapy: Potentiometric and Substrate Specificity Studies

E. coli nitroreductase A (NfsA) is a candidate for gene-directed prodrug cancer therapy using bioreductively activated nitroaromatic compounds (ArNO2). In this work, we determined the standard redox potential of FMN of NfsA to be −215 ± 5 mV at pH 7.0. FMN semiquinone was not formed during 5-deazaflavin-sensitized NfsA photoreduction. This determines the two-electron character of the reduction of ArNO2 and quinones (Q). In parallel, we characterized the oxidant specificity of NfsA with an emphasis on its structure. Except for negative outliers nitracrine and SN-36506, the reactivity of ArNO2 increases with their electron affinity (single-electron reduction potential, E17) and is unaffected by their lipophilicity and Van der Waals volume up to 386 Å. The reactivity of quinoidal oxidants is not clearly dependent on E17, but 2-hydroxy-1,4-naphthoquinones were identified as positive outliers and a number of compounds with diverse structures as negative outliers. 2-Hydroxy-1,4-naphthoquinones are characterized by the most positive reaction activation entropy and the negative outlier tetramethyl-1,4-benzoquinone by the most negative. Computer modelling data showed that the formation of H bonds with Arg15, Arg133, and Ser40, plays a major role in the binding of oxidants to reduced NfsA, while the role of the π–π interaction of their aromatic structures is less significant. Typically, the calculated hydride-transfer distances during ArNO2 reduction are smallwer than for Q. This explains the lower reactivity of quinones. Another factor that slows down the reduction is the presence of positively charged aliphatic substituents

Mechanism of Two-/Four-Electron Reduction of Nitroaromatics by Oxygen-Insensitive Nitroreductases: The Role of a Non-Enzymatic Reduction Step

Oxygen-insensitive NAD(P)H:nitroreductases (NR) reduce nitroaromatics (Ar-NO2) into hydroxylamines (Ar-NHOH) through nitroso (Ar-NO) intermediates. Ar-NO may be reduced both enzymatically and directly by reduced nicotinamide adenine dinucleotide or its phosphate NAD(P)H, however, it is unclear which process is predominant in catalysis of NRs. We found that E. coli NR-A (NfsA) oxidizes 2 mol of NADPH per mol of 2,4,6-trinitrotoluene (TNT) and 4 mol of NADPH per mol of tetryl. Addition of ascorbate, which reduces Ar-NO into Ar-NHOH, changes the stoichiometry NADPH/Ar-NO2 into 1:1 (TNT) and 2:1 (tetryl), and decreases the rate of NADPH oxidation. Ascorbate does not interfere with the oxidation of NADPH during reduction of quinones by NfsA. Our analysis of ascorbate inhibition patterns and both enzymatic and non-enzymatic reduction of nitrosobenzene suggests that direct reduction of Ar-NO by NADPH rather than enzymatic reduction is the predominant mechanism during nitroaromatic reduction