QUARTERLY Two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H nitroreductase: Description of quantitative

Enterobacter cloacae NAD(P)H:nitroreductase catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) M(-1) s(-1) to 10(7) M(-1) s(-1), and oxidizing 2 moles NADH per mole mononitrocompound. Oxidation of excess NADH by polynitrobenzenes including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of 'redox cycling' was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron- or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationships shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Enterobacter cloacae nitroreductase (NR) is a homodimeric 24.5 kDa protein containing an FMN cofactor which reduces nitrofurans, 2,4,6-trinitrotoluene (TNT), and other nitroaromatics [8].NR follows 'ping-pong' mechanism and reduces nitrobenzene to phenylhydroxylamine in two successive two-electron transfers at the expense of 2 molecules of NADH, the nitroso intermediate being reduced much faster than nitrobenzene [10].Thus, NR shares the properties of other oxygen-insensitive nitroreductases, such as Escherichia coli nitroreductase and DT-diaphorase [6][7][8].However, the structure-activity relationships and the mechanism of two-electron (hydride) transfer by E. cloacae NR have been insufficiently studied.These studies may be of certain interest, since various strains of Enterobacter cloacae are currently being used in biodegradation of nitroaromatic or nitrate ester explosives [11,12].
In the present work, we have studied the reactions of E. cloacae NR with a series of nitrocompounds including high explosives TNT, tetryl, pentryl, TNC, RDX, and HMX (Fig. 1) (cf.Abbreviations).Furthermore, we have established quantitative structure-activ- ity relationships linking the reaction rate with the enthalpies of single-and two-electron reduction of nitroaromatic compounds, obtained by quantum chemical calculations.
Enzymatic assays and analytical procedures.Kinetic measurements were carried out in 0.1 M Tris/Cl (pH 7.0) containing 0.5 mM desferrioxamine at 25°C.The rate of NR-catalyzed oxidation of NADH by various nitroaromatics was determined by monitoring NADH oxidation (De 340 = 6.2 mM -1 cm -1 ) using a Hitachi-557 spectrophotometer.Corrections were introduced when necessary for the formation of reaction products absorbing at 340 nm.The catalytic constant (k cat ) and the bimolecular rate constant (k cat /K m ) of nitrocompound reduction correspond to the reciprocal intercepts and slopes of plots where [E] is enzyme concentration, and [ArNO 2 ] is concentration of nitrocompound; k cat is the number of NADH molecules oxidized by a single active center of the enzyme per 1 s.The temperature dependence of k cat /K m of nitroaromatics was mea-sured between 15-45°C.The reduction of cytochrome c, added into the reaction mixture in separate experiments, was monitored spectrophotometrically using De 550 = 20 mM -1 cm -1 .The rate of oxygen consumption during enzymatic reactions was monitored using a Clark electrode.The concentrations of nitrite were determined spectrophotometrically as described previously [15].

RESULTS AND DISCUSSION
In accordance with previous observations [10], NR catalyzed the oxidation of 2 moles NADH per mole of mononitrobenzenes.During the NR-catalyzed oxidation of NADH by polynitrobenzenes, the rapid oxidation of 2 NADH equivalents was followed by a second slower phase of oxidation of more than 4 NADH equivalents (data not shown).NR participated in all phases of reaction, since both fast and slow phases were suppressed by 20 mM dicumarol.The first rapid phase of NR-catalyzed NADH oxidation by tetryl, pentryl, TNT or dinitrobenzenes was not accompanied by O 2 uptake or reduction of added cytochrome c, or the previously observed nitrite formation, characteristic of the reduction of tetryl and other nitroaromatic N-methylnitramines by DT-diaphorase [15].O 2 was consumed on a time scale of the subsequent phase, with rates and amounts nonstoichiometric to NADH oxidation (not shown).The addition of catalase caused reappearance of oxygen, indicating that H 2 O 2 was formed as a final reaction product.The second phase of reaction was also accompanied by the reduction of added cytochrome c, which was not inhibited by superoxide dismutase (60 mg/ml) (not shown).Evidently, the hydroxylamine products of reduction of polynitroaromatics were further reduced to dihydroxylamines or other products, which were subsequently reoxidized by O 2 with the formation of H 2 O 2 [7,21], and were able directly to reduce cytochrome c [22].During the second phase of reaction, tetryl, pentryl, and 2,4-dinitrophenyl-N-methylnitramine formed a stoichiometric amount of nitrite.The reduction by TNT was not accompanied by nitrite formation.However, characterization of the reaction products is beyond the scope of the present work.
In agreement with the 'ping-pong' scheme for the steady-state kinetics of E. cloacae NR [10], we have obtained a series of parallel plots in Lineweaver-Burk coordinates at varied concentrations of tetryl, pentryl or 2,4-dinitrophenyl-N-methylnitramine as electron acceptor (10-100 mM) and fixed concentrations of NADH (50-250 mM), (not shown).The values of k cat obtained by extrapolation to an infinite concentration of NADH and the above electron acceptors, and k cat /K m values of all the electron acceptors investigated are given in Table 1, together with the values of their single-electron reduction potentials (E 1  7 ).The kinetic parameters refer to the first phase of enzymatic reaction.The k cat /K m for NADH was almost identical for several electron acceptors used (tetryl, pentryl, and 2,4-dinitro-N-methylnitramine), being equal to (5.9 ± 0.77)´10 6 M -1 s -1 .
Similar to earlier data [8], the reactivity of E. cloacae NR increased upon increase in E 1  7 of several nitroaromatic compounds (Table 1).However, the linear correlation between log k cat /K m and E 1  7 values was poor, r 2 = 0.5227 (not shown), and the data were better described by parabolic approximation (r 2 = 0.7040) (Fig. 2).This indicates that the reactivities of nitroaromatics are determined mainly by their electron accepting properties, and not by their particular structure.This served as a starting point for the description of quantitative structure-activity relationships involving compounds with unknown E 1   7   values.It is known that the enthalpies of reactions (DHf) obtained by means of quantummechanical calculation, exhibit a correlation with single-electron transfer redox potentials [24] in ArNO 2 reduction is hydride transfer, yielding an anionic N,N-dihydroxylamine form (ArN(OH)O -) (Eqn.5): An analogous mechanism has been postulated for the nonenzymatic NADH reduction of quinones, where the rate-limiting net hydride transfer with the formation of anionic hydroquinone (QH -) is followed by fast protonation (formation of QH 2 ) [25].The calculated DHf values for all the possible steps in the two-electron reduction pathway (Eqn.5) are given in Table 2. Irrespective of the favourable energetics of reduction of nitroalicyclic compounds RDX and HMX (Table 2), their reactivities were negligible (Table 1).In spite of the evident specificity of NR to nitroaromatic compounds, the reactivities of RDX and HMX were not used in further structure-activity relationships.The linear correlation between log k cat /K m of nitroaromatics and DHf(ArNO 2 -. ) was characterized by r 2 = 0.7120 (PM3), and 0.6958 (AM1); however, the data were better described by a parabolic correlation (r 2 = 0.8106 (PM3) and 0.8495 (AM1, Fig. 3A)).The correlations between log k cat /K m and DHf for a net reaction (DHf(ArNO)) were poor: r 2 = 0.1090 (PM3) and 0.2877 (AM1) for a linear approximation, and r 2 = 0.5733 (PM3) and 0.5044 (AM1) for a parabolic approximation (data not shown).and 0.4589 (AM1) for a linear approximation, and r 2 = 0.6469 (PM3) and 0.7185 (AM1) for a parabolic approximation, data not shown).On the other hand, the use of DHf for hydride transfer alone DHf(ArN(OH)O -) resulted in linear correlations with r 2 = 0.7187 (PM3) and 0.7508 (AM1), and in even better parabolic correlations (r 2 = 0.8106 (PM3) and 0.8495 (AM1), Fig. 3B).Thus, among the calculated DHf values for all the possible steps in the two-electron reduction pathway (Eqn.5, Table 2), the use of DHf (ArN(OH)O -) resulted in the best approximation of the quantitative structure-activity relationship (Fig. 3B).This may indicate that the two-electron reduction of nitroaromatics by E. cloacae NR proceeds with a rate-limiting hydride transfer and that subsequent steps (i.e., protonation and dehydration, see Eqn. 5) are faster.
One should note that the rates of nitroaromatics reduction by NR depend equally well on both DHf of hydride and electron transfer (Fig. 3A, B), and increase upon an increase in their E 1  7 values (Fig. 2).This closely resembles the regularities observed in the reduction of quinones by 1,4-dihydronicotinamides: i) the parabolic reactivity vs. E 1 relationship and the transient formation of an ion-radical pair or charge-transfer complex [26] pointed to a multistep (e.g., e -, H + , e -) hydride transfer [26]; ii) the reaction rate increased upon an increase in redox potential of the quinone/anionic hydroquinone (Q/QH -)

946
H. Nivinskas and others 2000 ).The numbers of compounds are taken from Table 1.The numbers of compounds are taken from   couple as well, which was formally consistent with the single-step hydride transfer model [25].However, the possibility of multistep hydride transfer during reduction of nitroaromatics by NR requires additional lines of evidence, since the values of DS ¹ for nitroaromatics reduction obtained in the present work do not entirely favour this mechanism.For comparison, the multistep hydride transfer between dihydronicotinamides and quinones is characterized by a much more negative DS ¹ value (-134 J ´mol -1 ´K-1 [25]), a consequence of a large negative entropy of the transient charge-transfer complex formation.

CONCLUSIONS
In contrast to the well-documented reactivity vs. E Not determined in view of unavailable pK a values of free radicals and anionic N,N-dihydroxylamine reduction products of nitrobenzoic acids.enthalpies of two-electron (hydride) reduction obtained by quantum chemical calculations in the present work may serve as a useful tool for the analysis of the mechanisms of two-electron reduction, especially of nitroaromatic compounds with presently unknown redox potentials.Although the quantitative structureactivity relationships obtained in this work are specific for a particular enzyme, E. cloacae NR is a member of a larger family of proteins including the E. coli and Salmonella typhi-murium nitroreductases, with which it shares over 80% amino acid sequence identity [8,13].Thus, these results may be extended to related nitroreductases.
Enterobacter cloacae NAD(P)H : nitroreductase catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (k cat /K m ) been observed as a slower secondary process, accompanied by O 2 consumption.This type of 'redox cycling' was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products.The logs k cat /K m of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron-or two-electron (hydride) reduction, obtained by quantum mechanical calculations.This type of quantitative structure-activity relationships shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

942 H. Nivinskas and others 2000 Figure 1 .
Figure 1.The formulae of nitroaromatic and nitroalicyclic explosives studied in this work.
150 mM.The k cat values in parentheses were obtained at infinite concentrations of both substrates;

Figure 2 .
Figure 2. The dependence of log k cat /K m of nitroaromatic compounds on their single-electron reduction potentials (E 1 7 ).The numbers of compounds are taken from Table1.

Table 2 . Enthalpies of single-and two-electron reduction of nitroaromatics calculated by Eqns. 1-4 using PM3 and AM1 methods
DHf for reduction of 4-nitro group of polinitrobenzenes and 1-nitrogroup of tetranitrocarbazole.The DHf values for reduction of other nitro groups are more negative by 2-6 kJ/mol.
a b