Enzymatic Reduction of 2,4,6-Trinitrotoluene and Related Nitroarenes: Kinetics Linked to One-Electron Redox Potentials

Article abstract of DOI:10.1021/es991422f

At military bases and munitions factories 2,4,6-trinitrotoluene (TNT) is a common soil and groundwater contaminant. Although the reduction of nitro groups in TNT and related nitroarenes has been extensively investigated, few researchers have studied the l

Full text of DOI:10.1021/es991422f

Environ. Sci. Technol. 2000, 34, 3900-3906
monoamino isomers, 2-amino-4,6-dinitrotoluene (2ADNT)
Enzymatic Reduction of
and 4-amino-2,6-dinitrotoluene (4ADNT), and only one
diamino isomer, 2,4-diamino-6-nitrotoluene (DANT), form
in significant amounts from TNT reduction (11). As the
electron-withdrawing nitro groups are sequentially replaced
with electron-donating amino groups, the remaining nitro
groups become less susceptible to reduction. Thus, TNT is
reduced faster than ADNTs which, in turn, are reduced faster
than DANT (12). Complete anaerobic transformation of TNT
to TAT appears limited by the DANT to TAT transformation.
Many enzymes in both prokaryotic and eukaryotic cells
are capable of mediating TNT, ADNT, DANT, and DNT
reduction (13). These have been divided in type I nitro-
reductases that reduce in two-electron increments and type
II nitroreductases that reduce in one-electron increments.
These enzymes have other physiological roles but also exhibit
activity with nitro groups (13). Type II nitroreductases are
oxygen sensitive because the nitro anion radical formed after
one-electron reduction can react with oxygen, resulting in
electron transfer to oxygen yielding a superoxide radical and
the original nitro group (9). Most nitroreductases purified
from bacteria are type I soluble flavoproteins that use
nicotinamide adenine dinucleotide (NADH‚H+) or nico-
tinamide adenine dinucleotide phosphate (NADPH‚H+) as
electron donors (13). In redox reactions, these compounds
accommodate the transfer of two protons and two electrons
to yield NAD+ and NADP+, respectively. Many purified
enzymes and cell-free extracts exhibit activity against TNT
using either NADH‚H+ or NADPH‚H+ as electron donors
including enzymes from Pseudomonas pseudoalcaligenes(14),
P. aeruginosa (15), Bacillus sp. (15), Staphylococcus sp. (15),
Ralstonia eutropha (16), Enterobacter cloacae (17, 18), and
Clostridium thermoaceticum (19).
In several abiotic reduction studies, the rate of reduction
of nitroarenes and quinones was linked to the one-electron
redox potentials of the compounds (17, 20-26). Log-linear
relationships were observed between the reduction rates and
the one-electron redox potentials of the nitroarenes or
quinones. None of these studies, however, investigated the
compounds of interest here, namely, TNT, 24DNT, 26DNT,
4ADNT, 2ADNT, and DANT, because the one-electron
reduction potentials for these compounds had not been
determined. One-electron redox potentials have since been
estimated for 24DNT from a linear free energy relationship
(LFER) established with 10 nitroarenes with known one-
electron redox potentials (nitrobenzene, methylnitroben-
zenes, chloronitrobenzenes, and acetylnitrobenzenes) (22)
and for TNT, 2ADNT, 4ADNT, and DANT from a LFER
established with five nitroarenes (4-methylnitrobenzene,
4-chloronitrotoluene, 4-acetylnitrotoluene, 1,3-dinitroben-
zene, and 1,4-dinitrobenzene) (26).
2,4,6-Trinitrotoluene and Related
Nitroarenes: Kinetics Linked to
One-Electron Redox Potentials
R . G U Y R I E F L E R † A N D
B A R T H F . S M E T S * , † , ‡
Environmental Engineering Program, Department of Civil
and Environmental Engineering, and Microbiology Program,
Department of Molecular and Cell Biology, University of
Connecticut, Storrs, Connecticut 06269-2037
At military bases and munitions factories, 2,4,6-trinitrotoluene
(TNT) is a common soil and groundwater contaminant.
Although the reduction of nitro groups in TNT and related
nitroarenes has been extensively investigated, few
researchers have studied the link between reduction
rates and the electrochemical properties of these compounds.
In this work, the standard one-electron redox potentials
at pH 7 (E °′) for six important nitroarenes have been measured
1
by pulse radiolysis. The internally consistent values
were -0.253 V for TNT, -0.417 V for 2-amino-4,6-
dinitrotoluene, -0.449 V for 4-amino-2,6-dinitrotoluene,
-0.397 V for 2,4-dinitrotoluene, -0.402 V for 2,6-dinitrotoluene,
and -0.502 V for 2,4-diamino-6-nitrotoluene. The reduction
kinetics of these nitroarenes was investigated using a
bacterial nitroreductase, NAD(P)H:FMN oxidoreductase that
uses NADH‚H+ as a cosubstrate. A log-linear relationship
was observed between the E °′ values and the enzymatic
1
reduction rates for five nitroarenes, suggesting that transfer
of the first electron is the rate-limiting step in nitroreduction.
Introduction
2,4,6-Trinitrotoluene (TNT), a widely used explosive, is a
common contaminant at military bases and munitions
production and handling facilities. In addition to its explosion
hazard, TNT causes red blood cell abnormalities, liver
dysfunction, and cancer in mammals (1, 2). TNT is found as
a pollutant in soil and groundwater often in combination
with 2,4-dinitrotoluene (24DNT) and 2,6-dinitrotoluene
(26DNT), both precursors in the TNT manufacturing process
(3). Although some bacterial cultures are capable of partial
mineralization of TNT (4-6), TNT is most commonly
biotransformed by reduction of the nitro groups (7, 8). By
the sequential addition of two electrons, a nitro group is
reduced to a nitroso, a hydroxylamino, and finally an amino
group (9). Through successive reductions of the three nitro
groups, many bacterial species sequentially transform TNT
to aminodinitrotoluene, diaminonitrotoluene, and finally
triaminotoluene (TAT) (9). However, in some bacterial
cultures hydroxylamino-substituted compounds persist with-
out production of amino-substituted compounds (10). Two
In summary, the reduction of TNT and related nitroarenes
has been extensively investigated; however, few researchers
have studied the link between reduction rates and the
electrochemical properties of these compounds. In this work,
it was hypothesized that the reduction rates of TNT, 2ADNT,
4ADNT, DANT, 24DNT, and 26DNT are correlated to their
one-electron redox potentials. A single enzyme, NAD(P)H:
FMN oxidoreductase isolated from Photobacterium fischeri,
was employed in this research. This enzyme’s physiological
role is the coupled oxidation of NAD(P)H‚H+ and reduction
of flavin mononucleotide (FMN), but it has been shown to
reduce TNT and other nitroarenes (27). The objectives of
this research were (i) to measure the one-electron redox
potentials for TNT, 2ADNT, 4ADNT, DANT, 24DNT, and
26DNT and (ii) to verify the hypothesis that the enzymatic
* Corresponding author e-mail:
barth.smets@uconn.edu;
phone: (860)486-2270; fax: (860)486-2298.
† Department of Civil and Environmental Engineering.
‡ Department of Molecular and Cell Biology.
9
3 9 0 0 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 18, 2000
10.1021/es991422f CCC: $19.00
2000 Am erican Chem ical Society
Published on Web 08/04/2000

reduction rates of these compounds are related to their one-
electron redox potentials.
Presence of 2-methyl-2-propanol ensured that after each
pulse, radicals produced by water radiolysis (H•, OH•) were
converted to (CH3)3COH•. The remaining solvated electrons
(eaq-) and (CH3)3COH• reacted with either Q or S to produce
Q•- or S•-. These two redox couples quickly attain equilibrium
with an equilibrium constant calculated by
Materials and Methods
Chem icals. TNT was purchased from Chemservice (West-
chester, PA). 2ADNT, 4ADNT, and DANT were purchased
from Accustandard Inc. (New Haven, CT); 24DNT, 26DNT,
duraquinone (DQ), anthraquinone-2-sulfonate (AQS), and
methyl viologen (MV) were purchased from Aldrich (Mil-
waukee, WI). NADH‚H+ and NAD+ were purchased from
Sigma (St. Louis, MO), and Photobacterium fischeri NAD(P)H:
FMN oxidoreductase was purchased from Roche (Indian-
apolis, IN). Neat 2-hydroxylamino-4,6-dinitrotoluene, 4-hy-
droxylamino-2,6-dinitrotolune, and 2,4-dihydroxylamino-6-
nitrotoluene were generously provided by Dr. Joseph Hughes
(Rice University, Houston, TX) for identification of TNT
reduction products. TNT, 2ADNT, 4ADNT, DANT, and NADH‚
H+ were 99% pure, and NAD+ was 98% pure. Nitroarenes
were dissolved in deionized water (DI) and autoclaved at 121
°C to create stock solutions. Spectrophotometric and HPLC
analysis confirmed that no transformation occurred with this
treatment. NADH‚H+ and NAD+ stock solutions were pre-
pared in 50 mM tris(hydroxymethyl)aminomethane hydro-
chloride (TRIZMA) buffer at pH 7 and stored at 4 °C. NADH‚
H+ concentration was confirmed before each experiment by
spectrophotometric measurement. NAD(P)H:FMN oxido-
reductase was dissolved in 40% (by volume) glycerol con-
taining 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1
mM dithiothreitol, and 50 mM potassium phosphate buffer
at pH 7, per manufacturer instructions. The enzyme’s
reported activity was 100 U/ mg of protein at 25 °C and pH
7 with FMN and NADH‚H+ as substrates.
{Q•-}{S}
{S•-}{Q}
[Q•-][S]
[S•-][Q]
K )
) f
(1)
where {A} is the activity of A, [A] is the molar concentration
of A, and f is the composite activity coefficient for the four
dissolved compounds.
The quinone radical concentration is proportional to the
absorbance measured at the quinone radical peak wavelength
corrected for interference by nitroarene absorbance. Because
the electron pulse is highly reproducible, the same total
number of radicals is produced in each run. The concentra-
tion of nitroarene radicals is proportional to the decrease in
the absorbance at the quinone radical peak wavelength in
the solution when nitroarenes were present. In other words,
the difference in absorbance at the quinone radical peak of
a quinone-only experiment and a quinone-plus-nitroarene
experiment is proportional to the electrons transferred to
the nitroarene (28). Thus
[Q•-
[S•-]
]
A - A
s
)
(2)
Aq - A
where A, A , and A are the average absorbance at the quinone
s
q
radical peak wavelength of the test solution (quinone plus
nitroarene), the nitroarene-only solution, and the quinone-
only solution, respectively. Substituting eq 2 into eq 1 gives
Nitroarene concentrations were quantified using reversed-
phase HPLC with photodiode array detection (PU980 and
FP920 Jasco Inc., Easton, MD). Compounds were separated
on a Betasil Cyano column (Keystone Scientific Inc., Belle-
fonte, PA) using a methanol/ DI mobile phase (1 mL/ min)
buffered with 20 mM phosphate at pH 4, with a linear gradient
from 30% methanol to 45% methanol in 15 min. Absorbance
spectra from 200 to 400 nm (∆ ) 1 nm) were measured at
0.8-s intervals to ensure accurate identification of com-
pounds, while quantification was performed at 254 nm.
NADH‚H+ concentrations were determined by UV/ VIS spec-
trophotometry (Varian Cary Bio50, Sugar Land, TX) at 340
nm (ꢀNADH‚H+ ) 6,220 M-1 cm-1). NADH‚H+ strongly absorbs
at this wavelength, while NAD+ does not. The nitroarenes
absorb weakly at 340 nm, so concentrations of NADH‚H+
were kept 5 times higher than the nitroarene concentrations.
For experiments with TNT, 24DNT, 26DNT, 4ADNT, and
2ADNT paired with NADH‚H+, 2, 1, 1, 6, and 6%, respectively,
of the absorbance at 340 nm was due to interference from
the nitroarenes initially. Inspection of absorbance spectra of
nitroarene transformation products separated by HPLC
revealed insignificant change in the nitroarene-contributed
absorbance at 340 nm during the course of the experiments.
[S](A - A )
s
K ) f
(3)
[Q](Aq - A)
The one-electron redox potential in reference to the hydrogen
electrode (E1), at standard conditions (E1°), at pH 7 (E1°′) for
the nitroarenes can then be determined by
E1°′(S) ) E1°′(Q) - 0.059 log(K)
(4)
Activity coefficients are computed for the four dissolved
compounds using the Debye-Hu¨ ckel expression with unity
coefficients assumed for neutral species (32). At 5 mM
phosphate at pH 7, I ) 5.40 × 10-3 M. For nitroarenes and
•-
•-
DQ, activity coefficients are γS ) γQ ) 1 and γS ) γQ
)
•-
0.917. For AQS, which has a -1 charge, γQ ) 0.917 and γQ
) 0.708. For MV, which has a +2 charge, γQ ) 0.708 and γQ
•-
) 0.917. Thus, for solutions with DQ, f ) 1; with AQS, f )
0.842; and with MV, f ) 1.41.
Mixtures of a single nitroarene at concentrations ranging
from 0.01 to 0.2 mM and a quinone or methyl viologen at
concentrations ranging from 0.0001 to 1.0 mM were prepared
to yield concentration ratios, [S]/ [Q], from 0.1 to 200. After
preparation, the mixture was prepurged and continually
purged with N2 as the solution was discharged to the radiolysis
flow cell. Absorbance versus time profiles were averaged over
five pulses. At least three individual profiles were used at
each concentration ratio to determine average equilibrium
concentrations of quinone or methyl viologen radicals. Four
to six different concentration ratios were used to compute
a mean value and standard deviation of the equilibrium
coefficient. Mean and standard deviations for E1°′ were
computed from equilibrium coefficient estimates using eq
4 and appropriate error propagation formulas (33).
Because a fraction of the original compound was trans-
formed into radicals, [Q] and [S] were not equivalent to the
Pulse Radiolysis. Pulse radiolysis was performed accord-
ing to established procedures (28, 29). All experiments were
conducted at the U.S. Department of Energy Radiation
Laboratory at Notre Dame University (South Bend, IN).
Briefly, a nitroarene (S) and a quinone or methyl viologen
(Q) were dissolved in a 2-methyl-2-propanol ((CH3)3COH)
solution (0.2 M) buffered at pH 7 with 5 mM phosphate. TNT
was paired with DQ; 2ADNT, 4ADNT, 24DNT, and 26DNT
were paired with AQS; and DANT was paired with MV.
Solutions were irradiated by 2.5-ns pulses of 2.8 MeV electrons
using the Titan Beta model TS-8/ 16-1 S electron linear
accelerator (30). Solution absorbance was recorded at the
wavelength of maximum absorbance for the quinone radical
(445 nm for DQ, 505 nm for AQS, and 395 nm for MV) (31).
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starting concentrations. Using a known standard, potassium
thiocyanate, it was determined that 3.47 × 10-6 M solvated
electrons were formed in each pulse experiment. Given that
[Q•-]/ [S•-] was computed with eq 2, and [Q•-] + [S•-] ) 3.47
× 10-6 M, values for [Q•-] and [S•-] were determined, and [Q]
and [S] were likewise computed. E1°′ calculations for the six
nitroarenes used the reported values E1°′(DQ) ) -0.260 V,
E1°′(AQS) ) -0.390 V, and E1°′(MV) ) -0.448 V (34).
TABLE 1. Measurement of E °′ (V) by Pulse Radiolysis and
1
Other Reported Estimates (Average ( SD)
redox-
nitro- coupled
reported
E °′ (V)
arene
compd
n
K
E °′ (V)
1
1
TNT
2ADNT AQS
4ADNT AQS
DANT
24DNT AQS
26DNT AQS
DQ
5
5
4
6
5
4
0.760 ( 0.040 -0.253 ( 0.022 -0.300a
2.91 ( 0.65 -0.417 ( 0.096 -0.390a
9.98 ( 1.38 -0.449 ( 0.060 -0.430a
8.36 ( 5.30 -0.502 ( 0.275 -0.515a
1.31 ( 0.17 -0.397 ( 0.057 -0.360b
Enzym atic Transform ation Experim ents. Enzymatic
transformation of the nitroarenes was performed in 3-mL
sealed quartz spectrosil cuvettes (Aldrich, Milwaukee, WI)
with solution volumes of 1.5 mL. A TRIZMA buffered solution
of NADH‚H+, nitroarene, and enzyme was mixed in a cuvette
kept at 25 °C in a water-jacketed cuvette holder. To exclude
oxygen, the solution was initially sparged with water vapor
saturated N2 for 5 min, while N2 flush of the headspace
continued throughout the experiment. Cap septa were
replaced on a daily basis to maintain airtight seals. Initially,
control experiments were conducted to verify the absence
of transformations in solutions containing mixtures of
enzym e/ NADH‚H+, enzym e/ TNT, and TNT/ NADH‚H+.
NADH‚H+ and TNT were at 500 and 100 µM, respectively,
and the enzyme was at 0.533 mg of protein/ L. NADH‚H+ and
TNT concentrations were measured by absorbance at 340
and 254 nm, respectively.
MV
1.62 ( 0.37 -0.402 ( 0.098
nac
a
b
c
From ref 26. From ref 22. na, no estim ate available.
o
of the first electron transfer, ∆G1 ′:
o
∆Gq ) R∆G1 ′ + â
(6)
where R and â are constants (36). Finally, the free energy of
one-electron transfer can be related to E1°′ using the Faraday
constant, F:
o
∆G1 ′ ) F(E1°′(oxidant) - E1°′(reductant))
(7)
Combining these three relationships, using T ) 25 °C, and
using a constant value for E1°′(reductant) results in the linear
free energy relationship
An additional control experiment was conducted to
confirm that NADH‚H+ depletion was an accurate measure
for nitroarene depletion. Enzymatic 2ADNT transformation
was used as a worst-case scenario, because 2ADNT has the
highest interference with NADH‚H+ absorbance at 340 nm.
The experiment was initiated by adding the enzyme at 7.5
mg of protein/ L to a solution of 2ADNT and NADH‚H+ at 100
and 500 µM, respectively. NADH‚H+ consumption was
monitored spectrophotometrically, and periodic samples
were removed for HPLC analysis.
E1°′(oxidant)
log k ) A
+ B
(8)
0.059
where A and B are constants (36). Values for A and B were
determined by least-squares regression and minimizing
absolute deviations (MAD) (37).
The Marcus theory of outer-sphere electron transfer was
used to interpret A values obtained from eq 8. Marcus theory
assumes that a reversible electron-transfer reaction takes
the form
For reaction rate measurements, experiments were initi-
ated by adding the enzyme to solutions that contained NADH‚
H+ and a single nitroarene at 500 and 100 µM, respectively.
NADH‚H+ consumption was monitored by spectrophotom-
etry. An enzyme aliquot was injected into the capped cuvette
through the septum, and the solution was rapidly mixed
before continuing spectrophotometric observations. Initial
and final aliquots were collected for HPLC analysis to
determine final concentrations of nitroarenes and to identify
transformation products. NADH‚H+ consumption profiles
were translated into nitroarene reduction profiles using the
stoichiometry calculated from measured initial and final
substrate concentrations. Experiments were conducted with
TNT, 2ADNT, 4ADNT, DANT, 24DNT, and 26DNT. Initial
reaction rates were determined by estimating the slope of
the initial profiles of nitroarene consumption (e2 min) using
standard linear regression. Rates were normalized with
respect to the enzyme and initial substrate concentrations.
If an initial linear region was not observed, the experiment
was repeated with a different enzyme concentration until a
clear initial linear region was observed. Experiments were
conducted at several different initial enzyme concentrations.
kd
kel
•-
A + D S (AD) S (A D•+) w products
(9)
k-d
-kel
where kd is the rate constant for complex formation, k-d is
the rate constant for complex disassociation, kel is the rate
constant for electron transfer, and k-el is the rate constant
for back electron transfer (35). The Marcus equation relates
the observed overall reaction rate, kobs, and the pH-corrected
standard Gibbs free energy of the electron-transfer step, ∆G1°′.
Three significant scenarios are observed: (i) a diffusion-
limited case where kobs is controlled by kd; (ii) an activation-
limited case where kobs is controlled by kel; and (iii) an
equilibrium-controlled case where k-el is significant (35).
When the reaction is equilibrium controlled (case iii), the
slope of log k versus ∆G1°′ is -1/ 2.3RT, the slope of log k
versus E1°′ is nF/ 2.3RT, and the slope of log k versus E1°′/
0.059 V is 1. When the slope of log k versus E1°′/ 0.059 V is
near 0, the overall reaction rate is controlled by the rate of
precursor formation (case i). Slope values between 1 and 0
may indicate electron-transfer control (case ii).
Transition state theory was used to relate reaction rates
with E1°′. For reactions that have a common rate-limiting
step, a correlation between the free energy of activation for
that rate-limiting step, ∆Gq, and the reaction rate, k, is often
observed (35):
Results
Pulse Radiolysis. Average and standard deviations of ex-
perimental Kand E1°′ values for TNT, 2ADNT, 4ADNT, 24DNT,
26DNT, and DANT are reported in Table 1. Calculated values
of K varied little over an order of magnitude range of
concentration ratios for all chemicals except DANT, which
had a coefficient of variation (CV) of 63.4%. E1°′ values varied
from -0.253 for TNT to -0.502 for DANT. As with K, much
greater uncertainty was observed in the E1°′ estimate of DANT,
∆Gq ) -2.3RT log k + c
(5)
where R is the universal gas constant, T is the absolute
temperature, and c is a constant. Additionally, the free energy
of activation is often proportional to the free energy change
9
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FIGURE 1. 2ADNT depletion profile during an enzyme assay calculated from NADH‚H+ depletion (+) and measured directly via HPLC (0).
CV of 54.8%, than of the remaining compounds, CVs of 8.7-
TABLE 2. Average Initial Reaction Rates for the Reduction of
24%. Maximum precision was achieved with TNT, CV of 8.7%.
Five Nitroarenes (Average ( SD)
Enzym atic Experim ents. Initially, control experiments
were performed to confirm that NADH‚H+, TNT, and enzyme
range of
enzyme concn
(mg of protein/L)
normalized reaction rate
were all necessary components for reaction. When only two
of the three components were mixed, no activity was observed
during the time scale of the experiment, indicating that no
unexpected oxidation/ reduction reactions occurred in this
system.
compd
n
(mL (mg of protein)-1 min-1
)
TNT
8
2
3
2
3
0.133-0.533
4.00, 5.33
5.33-8.00
6.67
594 ( 104
91.1 ( 24.5
26.1 ( 14.6
21.0 ( 1.4
10.8 ( 4.9
24DNT
26DNT
2ADNT
4ADNT
An experiment was conducted to validate the use of an
NADH‚H+ consumption profile for calculating a nitroarene
consumption profile. NADH‚H+ and 2ADNT were concur-
rently measured during an enzymatic reaction by UV/ VIS
spectrometry and HPLC, respectively. After 85 min, 91.1 µM
2ADNT and 178 µM NADH‚H+ were transformed, resulting
in a stoichiometric ratio of 1.95 mol of NADH‚H+/ mol of
2ADNT. Three moles of NADH‚H+ are required for reduction
of a nitro group to an amino group, while a 2:1 ratio is indic-
ative of reduction to a hydroxylamino group. HPLC chro-
matograms revealed two peaks that did not match the elution
time or spectra for DANT. These peaks presumably were
2-amino-4-hydroxylamino-6-nitrotoluene and 2-amino-6-
hydroxylamino-4-nitrotoluene, although confirmatory stand-
ards could not be obtained. Assays with TNT, for which
appropriate standards were available, resulted in the forma-
tion of 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxyl-
amino-2,6-dinitrotoluene without formation of 2ADNT or
4ADNT. These results indicate that this enzyme transforms
nitro groups to the hydroxylamino level, not the amino level,
as has been observed in other bacterial systems (10).
The 2ADNT consumption curve was calculated from
NADH‚H+ consumption data using the calculated 1.95
stoichiometric ratio. The HPLC measured 2ADNT values
match the calculated values well, indicating a consistent
stoichiometry between the two reactants (Figure 1). The large
number of high-quality data points obtained by absorbance
measurements, particularly at the beginning of the experi-
ment, illustrates the advantage of measuring NADH‚H+
instead of the nitroarene and facilitates measuring initial
reaction rates. Furthermore, over the considered concentra-
tion ranges, standard deviations of the HPLC (3.27 µM
2ADNT) and spectrophotometric measurements (0.0283 ABU
corresponding to 2.60 µM 2ADNT) were very close.
6.67-13.3
a range of initial conditions by varying the initial enzyme
concentration. Normalized reaction rates were not impacted
by the initial enzyme concentrations (data not shown). TNT
was reduced most rapidly, followed by 24DNT, 26DNT,
2ADNT, and 4ADNT. No transformation of DANT was
observed with as much as 26.7 mg of protein/ L of enzyme.
This trend of decreasing activity coincides with the trend of
decreasing one-electron redox potentials (Table 1). Log
reaction rate was plotted versus E1°′/ 0.059 for the five
nitroarenes tested (Figure 2). Kinetics of TNT, 26DNT, 2ADNT,
and 4ADNT reduction obeyed a linear free energy relationship
(eq 8) quite well (Figure 2). 24DNT however deviated from
this relationship, i.e., 24DNT was reduced faster than
predicted by its E1°′. The best-fit linear regression equation
for the five pairs is
0.509E1°′
log k )
+ 5.01
(10)
0.059
with an R 2 ) 0.906 and standard deviations for A and B of
0.095 and 0.11, respectively. This fit is disproportionately
influenced by the TNT result because the E1°′/ 0.059 for that
data point is much higher than the remaining data points.
Methods to reduce the influence from this point include
removing the outlier or using a fitting method that puts less
relative weight on outliers (37). Removing the TNT data point
greatly changes the resulting linear fit because of greater
influence from the 24DNT data point (A ) 0.829, B ) 7.28).
Because the TNT data point has the greatest reliability (i.e.,
smallest standard deviation), it should be retained in the fit.
The MAD, which puts less relative weight on outliers, was
employed, and very similar regression results were obtained
(A ) 0.524, B ) 5.02).
Experiments were performed to determine the transfor-
mation rates of TNT, 2ADNT, 4ADNT, 24DNT, 26DNT, and
DANT (Table 2). Average reaction rates were determined over
9
VOL. 34, NO. 18, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 3 9 0 3

FIGURE 2. Average-normalized initial reaction rate as a function of E °′ for five nitroarenes and best-fit linear regression. The 95%
1
confidence interval for the linear regression is shown, and error bars indicate 1 SD.
TABLE 3. Linear Free Energy Relationships between Nitro Reduction Rates and E °′
1
a
rate
catalyst
A
ref
first-order reaction rate
initial reaction rate
first-order reaction rate
initial reaction rate
initial reaction rate
Vm ax/Km
m agnetite/Fe2+
cytochrom e P-450 reductase
hydrogen sulfide
Enterobacter cloacae nitroreductase
NAD(P)H:FMN oxidoreductase
flavocytochrom e b2
adrenodoxin
iron(II) porphyrin
iron(II) porphin
cytochrom e P-450 reductase
ferredoxin oxidoreductase
cytochrom e P-450 reductase
Streptom yces exudates
2-hydroxy-1,4-naphthoquinone
8-hydroxy-1,4-naphthoquinone
8-hydroxy-1,4-naphthoquinone
8-hydroxy-1,4-naphthoquinone
0.22-0.42
0.360
0.45-1.0
0.475
0.509
0.581
0.599
0.60
0.60-0.93
0.677
0.820
0.885
0.985
0.99
25
23
22
17
this research
24
24
25
21
24
20
20
41
21
21
22
26
Vm ax/Km
first-order reaction rate
first-order reaction rate
V
V
V
m ax/Km
m ax/Km
m ax/Km
first-order reaction rate
first-order reaction rate
first-order reaction rate
first-order reaction rate
first-order reaction rate
1.00
1.0
1.25
a
Param eter from the linear regression equation log k ) AE1°′/0.059 + B.
of the dissolved compound to support localized charge on
a nitro group (39). Nitro groups ortho to the methyl group
are shielded from stabilizing solvent effects by the hydro-
phobic methyl group, thus resisting polarization (39). Again,
the result is lower stability of the one-electron reduced 4ADNT
species than the one-electron reduced 2ADNT species.
Consistent with these explanations, increased reduction rates
for 2ADNT over 4ADNT have been observed (40) (Table 2).
The same trends of lower E1°′ values for compounds with
only ortho nitro groups are observed for the DNT isomers.
Again, the results presented here are consistent with trends
expected from the respective group contributions; however,
the difference in E1°′ values for the pairs 2ADNT/ 4ADNT and
24DNT/ 26DNT are not statistically significantly.
Discussion
Qualitative relationships between aromatic nucleus substit-
uents and one-electron redox potentials of the nitroarenes
are apparent (38) (Table 1). Comparing TNT with 4ADNT
and 2ADNT, as electron-withdrawing nitro groups are
exchanged with electron-donating amino groups, the electron
densities of remaining nitro groups increase. Thus, it is more
difficult to reduce the remaining nitro groups, and the E1°′
decreases. A similar trend is observed comparing 4ADNT
and 2ADNT with DANT and comparing TNT with 24DNT
and 26DNTselectron-withdrawing nitro groups are ex-
changed with electron-donating hydrogen groups. Also,
2ADNT and 4ADNT have lower E1°′ than 24DNT and 26DNT
because the amino group is a stronger electron donor than
the hydrogen group. Thus, all six results are consistent with
trends expected from the respective group contributions.
Comparing the aminodinitrotoluene isomers, 4ADNT has
a more negative E1°′ value than 2ADNT. This may be explained
by both steric and solvent effects. The nitro groups ortho to
the methyl group likely experience steric hindrance forcing
the resonance structures out of the benzene ring plane (38,
39). The result is lower stability of the one-electron reduced
4ADNT species than the one-electron reduced 2ADNT species
(38). Alternatively, reduction may be enhanced by the ability
To our knowledge, only two studies have previously
published values of E1°′ for TNT, ADNTs, DANT, or DNTs
(22, 26). In both of these studies, E1°′ values were inferred
from LFERs between the reduction rate of a suite of
nitroarenes and 8-hydroxyl-1,4-naphthoquinone (jugalone).
The reduction rates of TNT, 2ADNT, 4ADNT, DANT, and
24DNT with jugalone were measured, and E1°′ values were
estimated from the LFER. To develop the LFER, Dunnivant
et al. (22) used nitrobenzene, 2-nitrotoluene (NT), 3NT, 4NT,
2-chloronitrobenzene (CNB), 3CNB, 4CNB, 2-acetylnitroben-
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3 9 0 4 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 18, 2000

zene (ANB), 3ANB, and 4ANB. The E1°′ value for 24DNT we
determined via pulse radiolysis and the one computed from
the LFER were only 9.3% different (Table 1). Hofstetter et al.
used 4NT, 4CNB, 4ANB, 1,3-dinitrobenzene, and 1,4-dinitro-
benzene to develop an excellent log-linear LFER fit (26). The
E1°′ values for 2ADNT, 4ADNT, and DANT from our pulse
radiolysis experiments and the LFER were in close agreement
(6.5, 4.2, and 2.6% difference for 2ADNT, 4ADNT, and DANT,
respectively; Table 1). The E1°′ value we measured for TNT
deviated by 19% from the value reported by Hofstetter et al.
(26). These authors supplied electrons for TNT reduction by
a H2S/ jugalone system. Because the reduction rate of TNT
was 2-4 orders of magnitude faster than for ADNT and DANT,
it is possible that the reduction rate was limited by the
transport of electrons from H2S via jugalone to TNT rather
than the kinetics of the one-electron transfer, leading to an
erroneous low estimate for E1°′. We believe that our meas-
urement for E1°′ may be more accurate because the method
is more direct. Except for TNT, E1°′ results presented in this
study are very consistent with previously reported values.
Acknowledgments
We thank the U.S. Department of Energy Radiation Labora-
tory at the University of Notre Dame, especially Dr. Dan
Meisel and Tim Schatz, for operation of the linear electron
accelerator and assistance in the E1°′ measurements.
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9
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Received for review December 19, 1999. Revised manuscript
received June 14, 2000. Accepted June 19, 2000.
ES991422F
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