Term paper, June 2007

Mechanisms of oxidative vs. reductive nitrobenzene transformation by microorganisms

Term paper in Biogeochemistry and Pollutant Dynamics, Master Studies in Environmental Sciences, ETH Zrich By Ivo Caravatti Tutor: Dr. Thomas Hofstetter

Abstract
Nitroaromatic compounds such as nitrobenzene (NB), are widespread environmental contaminants. In natural attenuation processes selected microorganisms are able to degrade nitrobenzene via two different pathways, that is oxidation of nitrobenzene to catechol and reduction to aniline, respectively. Since both processes occur under oxic conditions but the latter leads to products of similar toxicity than the initial contaminant, it is necessary to differentiate between the two reactions at contaminated sites. Using compound-specific isotope analysis (CSIA), it should, in principle possible to identify NB degradation pathways by measuring changes of stable isotope signature of the remaining NB during its degradation. This hypothesis relies on the different enzymatic mechanisms that have been reported for enzymatic nitrobenzene degradation. However, since data on isotope effect are lacking, this term paper reviews the elementary reaction steps of the competing enzymatic nitrobenzene transformation pathways on a molecular scale to obtain some estimates of the potential magnitude of carbon and nitrogen isotope effects. Oxidation of nitrobenzene by Comamonas sp.strain JS765, should result in a moderate C isotope effect. The magnitude of the isotope effect, however, depends on some elementary reaction steps in the dioxygenation mechanisms, which are not fully understood to date. In addition, depending on the reaction mechanisms, also a strong N isotope effect might be expected. On the other hand, reduction of NB by Pseudomonas pseudoalcaligenes strain JS45 is expected to result in negligible C and substantial N isotope fractionation. The theoretical evidence for isotope fractionation obtained in this term paper suggest that the two enzymatic nitrobenzene transformation pathways might be distinguished in the field on the basis of compound-specific isotope analysis.

1. Introduction
Nitroaromatic compounds (NACs) constitute a very important class of environmental contaminants. The stability of nitroaromatic compounds make them valuable as chemical intermediates in industrial chemistry (8). Nitroaromatic compounds are used especially for the production of pesticides, dyes, and polymers. On the other hand, nitroaromatic explosives, which are derived from nitration of toluene, constitute a big problem at sites of military explosive production, ammunition testing and storage. The widespread application NACs and their improper disposal has resulted in the release of NACs into the environment (10). Innumerable sites are contaminated worldwide. In 1

Term paper, June 2007 addition, NACs used as agrochemicals such as pesticides are spread deliberately into the environment (9). Fortunately, many nitroaromatic compounds accumulated in the environment are biodegraded by bacteria. Bioremediation can be defined as any process that uses microorganisms to return a polluted environment to its original conditions. Adding specific substrate is possible to induce or accelerate a determinate biodegradation process. This process is called stimulation. Since mechanical cleanup of nitroaromatic polluted areas is nowadays an economic issue, bioremediation is an important alternative (9). However, compounds like nitrobenzene (NB) have more than one biodegradation pathway. Comamonas sp.strain JS765 is able to oxidise NB, on the other hand Pseudomonas pseudoalcaligenes strain JS45 is able to reduce NB (14, 15). Fig 1 shows the main step of the NB degradation for both microorganisms. Although both degradation pathways lead to mineralization of the compound, some products of the reductive pathway are transient more toxic products (especially aniline) Many of this products are cytotoxic and/or mutagenic (20).

NO 2 A) Oxydation B) Reduction

OH Catechol OH

NH2 Aniline

CO2 + NO 2-

CO2 + NH3

Figure 1: Main steps of the degradation of NB: A) Oxidation leads to the formation of catechol; B) Reduction leads to the formation of transient more toxic products. Both pathways lead to the complete degradation of NB. Modified from (14, 15).

It is important to determine which pathway occurs in the contaminated site. However, a direct measurement of the biodegradation products is labour intensive and not always conclusive for the distinction of the pathways. An increasingly important tool for qualitative and quantitative assessment of abiotic and enzymatic transformations of organic contaminants in the environment is the Compound Specific Isotope Analysis (CSIA)(6). CSIA is based on isotopic signature. Isotopic signature is determined through the relative isotopic compositions of a given element within a compound. Isotope signatures for a given element E is defined as follow (6):

Term paper, June 2007

# & & hE # ! $$l ! $ ! E ! $% " h E = $ h sample 1! 1000 E# ! $& $ ! l $ ! ! $ E % % " reference "

(1)

As isotope signature values are rather small, the number is converted into 0/00. hE and lE represent the relative abundance of heavy and light isotope of element E, respectively, in the sample and in an international reference standard (4). If isotope signatures are studied on more than one element, information on specific reaction mechanisms can be derived. As during the course of a reaction (e.g. a bond breaking or formation) the isotopes of a given element E present in the substrate do not react with the same rate constant, one of the isotope can be enriched in the substrate. This effect is called Kinetic Isotopic Effect (KIEE). Generally, the light isotope reacts faster than the heavy one, so that the heavy isotope is enriched in the substrate. Normally, this enrichment is independent of the concentration of the compound; therefore an enrichment factor for the element E (!E) can be calculated, !E is commonly reported in per mil ()(6):

& 1000 + h E ln$ $ 1000 + h E 0 %

# E ! ! = 1000 ln f "

(2)

Where f is the fraction of compound that has not reacted (usually c(t)/c(t=0)), hE and hE0 are the isotopic signatures of the compound for the element E at times t and zero, respectively. As explained above changes in isotope signature ("#E) are due to different reaction kinetics of the isotopes. !E-values can dependent on environmental conditions. Considering the reaction mechanisms it is possible to calculate an apparent kinetic isotope effect (AKIEE). This can be directly derived from the experimentally observed !E. AKIEE can be calculated as follow (6):

AKIE E =

1 k obs = h k obs 1 + E 1000

(3)

If only one atom of the element E is present in the molecule and this atom is taking part to the reaction, $ is equal 1. If several atoms of the same element are present in a contaminant, those not taking part in the reaction will decrease the observable isotope fractionation and the corresponding !E-value due to isotopic dilution (7). It is important to know the reaction mechanisms so that the element involved in the different biodegradation reactions can be defined. Bond cleavage or creation have different effects on KIE and therefore on !E. Enrichment factors are directly dependent on reaction mechanisms (4). In addition, it is important to distinguish between primary isotope effects and secondary isotope effects. Primary isotope effects are observable on atoms involved in a bond cleavage or formation. Secondary isotope effects are observable on atoms that are not directly taking part to the cleavage or formation reaction. Normally primary isotope effects are bigger than secondary. Hydrogen has a big weight difference between its isotopes, so that often secondary isotope effects for this atom are experimentally accessible (4).

Term paper, June 2007 Unfortunately, the exact values for the kinetic isotope effect for NB degradation reactions are not known yet. As point out above, the products of the NB biodegradation are not always conclusive for the distinction of the pathways; one of the advantages of using CSIA is that you measure only the change in isotopic signature in the substrate (NB).

2. Goals
Goal of this term paper is to asses the magnitude of isotope fractionation of the two alternative NB degradation pathways that is reductive and oxidative. This means to discuss the main elementary reactions steps that lead to a substantial isotope effect. In the following chapters, I review the elementary reaction steps, for NB oxidation and reduction, where bond are broken or bonding between the elements is changed significantly. In other words, this means that I try to identify where elementary reaction steps can cause a primary isotope effect. Since secondary isotope effects are usually one order of magnitude smaller and are more difficult to exploit for to the identification of isotope fractionation (4). Transport processes shows a negligible isotope effect and therefore masks isotope effect caused by bond cleavage if transport is limiting. In this work, we assume that transport processes do not limit the kinetic of NB transformation. Thus, I discuss isotope effects that are linked to bond cleavage and formation. The results of this research can than be applied for the design isotope fractionation experiment in laboratory and field.

3. Results and Discussion

3.1. Oxidative Pathway
Comamonas sp.strain JS765 is able to oxidise NB: product of this reaction is catechol, whereas nitrite is released. Figure 2 gives an overview of the elementary reaction steps taken in consideration. First NB enter into the bacteria (1); second NB binds to the enzyme responsible for the oxidation (2). After the formation of the enzyme substrate complex, two atoms of oxygen are added (3), finally catechol is produced and nitrite is released (4). To understand how elementary reaction can influence isotopic enrichment factors and which kind of enrichment factors can be expected, a closer look at the processes influencing this reaction is necessary. Therefore one needs to understand enzymatic reaction.

Figure 2: overview of the elementary reaction steps occurring until nitrite is released (breaking of the C-N bond). E = NBDO (Nitrobenzene dioxygenase), S = NB, P = Catechol. Elementary reaction steps: 1) NB

Term paper, June 2007

enters in to the bacteria and it is transported to the reactive-site of NBDO (E); 2) NB bind to the active site of NBDO, the complex ES is built; 3) Dioxygenase reaction: two atoms of oxygen are added 4) Catechol is produced, Nitrite is released.

Unfortunately, less is known about the transport of NB into the bacteria as well as the transport to the active site. The rate constant of each elementary reaction step is important for determine isotope effects. The overall reaction rate for the dioxygenation is determined by the rate of the slowest step (18). In part 3.1.1. the effects of elementary reaction steps 3 and 4 on isotope fractionation are discussed in detail.

3.1.1. Enzyme description, active-site and substrate binding

In this chapter, a closer look to the elementary reaction step 2 in figure 2 will be taken. Nitrobenzene dioxygenase (NBDO) from Comamonas sp.strain JS765 is able to add two atoms of oxygen to NB. Two electron and two protons are consumed during this process. As result a dihydroxyl intermediate (EP in figure 2) is produced. Through spontaneous rearrangement a molecule of nitrite is released and a molecule of catechol (P in figure 2) is formed (5). NBDO from Comamonas sp.strain JS765 was molecularly characterised by Lesser et al.(2001) (13). Successful purification and characterisation of all enzyme components were reached by Parales et al (2005) (17). Based on gene order and degree of sequence similarity NBDO belongs to the naphthalene family of Rieske nonheme iron dioxygenases (RDOs) (13). Three main components, a reductase, a ferrodoxin and a dioxygenase, build the multicomponent enzyme system. NBDO (the dioxygenase component) is a hexamer composed of three % and three & subunits. The active site is in a % subunit. Figure 3 shows how the multicomponent enzyme system works. The dioxygenase reaction takes place only when the active-site iron is in the reduced form (5).

Figure 3: Electron transfer chain in a Rieske nonheme iron dioxygenase (RDO) enzyme system. (Modified from (2)) Both the ferrodoxin component and the dioxygenase component contain a [2Fe2s] iron sulfur cluster responsible for the electron transfer. The dioxygenase contain also an iron atom in the active-site

The active site, which has an oval-shaped form, is formed by seventeen amino acid residues. Most of them are hydrophobic and provide an appropriate environment for the binding of aromatic substrate (5). However only three residues distinguish the NBDO activity from the activity of the others RDOs. Those are: an aspargine whose amino group is able to form a hydrogen bond with the nitro groups of nitrobenzene, an isoleuctine and a phenylalanine which are important for the correct positioning of the substrate and the dimension of the active pocket (10, 12). The formation of the hydrogen bond is represented in figure 2 as ES after elementary reaction step 2.

Term paper, June 2007 NBDO has high similarity with other RDOs. For example naphthalene dioxygenase (NDO) has 80% of sequence identity with NBDO (5) the reductase and ferrodoxin components of NBDO are identical of this of 2-Nitrotoluene Dioxygenase (2-NTDO) (13). This explain the ability of NBDO to use as substrate compound such as naphthalene, 2-nitrotoluene, 3-nitrotoluene, 2, 4dinitrotoluene. Figure 4 shows the iron molecule in the active site and the hydrogen bond between NB and the amino group of aspargine.

Figure 4: in red, the iron at the active site coordinated by two histidines, one aspargine and two molecule of water (not shown). The amino group of the aspargine is able to form a hydrogen bond with the nitro group of NB (Modified from (5))

3.1.2. Reaction mechanism: dioxygenation and release of Nitrite

Here elementary reaction 3 and 4 in figure 2 are discussed. Boyd et al (2005) (2) investigated the possible mechanisms for the dioxygenation of aromatic compounds. Figure 5 shows the two possible dioxygenation pathways for NB. It is demonstrate that both oxygen atoms come from the same oxygen molecule, so that two consequent monooxygenation reactions are to exclude (15). In case A oxygen atoms are added sequently, while in case B both atoms are added simultaneously. It is not known which of the two mechanisms is used by the enzyme; however since it is suggested that case B might be the used one (2), here isotopic fractionation consideration are based on case B. During the reduction reaction a C-C double bond is transformed to a C-C single bond, two O-C bonds are formed and a C-N bond is broken. For the analysis of the relevant elementary reaction steps the time of the release of nitrite plays a central role. It is not clear yet if nitrite is released from the dihidroxyl intermediate (ES in figure 2) during the addition of oxygen (case 1) or after the oxygen addition (case 2). However, the fact that the release of nitrite is involved in the limiting step, supports the thesis that nitrite is released during the addition of oxygen (15). If we assume that the C-N bond is broken during the addition of oxygen (case 1) primary isotope effect will be measurable for carbon and for nitrogen. On the other hand, if nitrite is released after the addition of oxygen (case 2), primary isotope effect will be observable only for carbon. Indicative substantial AKIE values for the cleavage of a double C-C bond is 1.01. AKIE value for the cleavage of a C-N bond is 1.03 for both elements (carbon and nitrogen) (4). Important to say is that hydrogen might undergo to an important secondary isotope effect. Hydrogen is not directly involved in a bond cleavage or formation reaction. However due to the big

Term paper, June 2007 difference in the weight between the two hydrogen isotopes, even a small isotope effect might leads to measurable enrichment factor.

Figure 5: possible catalytic mechanisms for NB dioxygenation; case A: sequently addition of the O atoms, case B: simultaneously addition of the O atoms. In blue the dihidroxyl intermediate. (Modified from (2))

3.2 Reductive Pathway

Pseudomonas pseudoalcaligenes strain JS45 is able to reduce NB: a product of the reduction reaction is nitrosobenzene. As the formation of nitrosobenzene is the only step which cause a measurable isotope effect, only this reaction is here taken in consideration. Figure 6 shows the elementary reaction steps occurring until the first bond is broken (N-O bond). First NB enter into the bacteria (1); second NB binds to the enzyme responsible for the reduction (2). After the formation of the enzyme substrate complex, two electrons are added (3), finally nitrosobenzene is released (4) (14). In the same way as for the oxidative pathway, we assume that the rate limiting step is the step where a bond is broken. In this case elementary reaction steps 4. A primary isotope effect will so be observable only for two elements. Those are nitrogen and oxygen.

Term paper, June 2007

Figure 6: overview of the elementary reaction steps occurring until nitrosobenzene is produced (breaking of the N-O bond). E = nitrobenzene nitroreductase, S = NB, P = Nitrosobenzene Elementary reaction steps: 1) NB enters in to the bacteria and it is transported to the reactive-site of nitrobenzene nitroreductase; NB bind to the active site of nitrobenzene nitroreductase, the complex ES is built; 3) Reduction reaction: addition of two electrons; 4)The product nitrosobenzene is released.

The reaction can be summarized as follow: Ar-NO2 + 2e- +2H+ ! Ar-NO + H2O

3.2.1 Enzyme description: reaction mechanism

Nitobenzene nitroreductase was purified and characterized by Somerville et al.(1995)(20).The enzyme does not have metal cofactors. It was found that two FMN (Flavin mononucleotide) molecule are bonded to the protein as cofactor (20). The reduction reaction is NADH dependent (14). Nitrobenzene nitroreductase belongs to the oxygen insensitive (type 1) enzyme (20). This category of enzyme uses a two electrons reduction. In contrast oxygen sensitive enzyme (type 2) catalyses a one electron reduction of the nitro group: a nitro anion radical is built. The radical reacts than spontaneously with oxygen building a superoxyde radical so that the nitro group is regenerated and no net reduction of the nitro group is observable (19). A two electrons reduction reduces the nitro group irrespective of the presence of oxygen. To figure out the mechanisms of the NB reduction scientists studied the reduction of NB by NAD(P)H nitroreductase by Enterobacter cloacae. Unfortunately the mechanism of two electrons reduction of nitroaromatics is not enough understood to provide any information about isotope fractionation (16). The nitro group is a facile electron acceptor. Many enzymes are able to catalyse the reduction of aromatic nitro group. For example cytochrome c reductase, cytochrome P-450 reductase, glutathione reductase, hepatic NAD(P)H reductase and many others are able to catalyse the reduction. One should note that the physiological role of this enzyme is not the reduction of aromatic nitro group (20). Interestingly cytochrome P-450 reductase has analogously to nitrobenzene nitroreductase a FMN and a FAD (flavin adenin dinucleotide) molecules as cofactors. In fact both enzyme are flovoenzyme (11). NB is a substrate for cytochrome P-450 reductase (3). The proposed mechanisms for the reduction of NB for both enzyme is a ping pong mechanisms (1, 3, 11). Figure 7 represents a possible mechanism. For cytochrome P-450 reductase is suggested that electrons transfer follows the pathway NADH! FAD ! FMN ! electrons acceptor. Probably the binding-site for NADH is a cysteine residue (3). 8

Term paper, June 2007 During the reductive degradation pathway of NB, Nitrosobenzene reacts fast to the next metabolite hydroxylaminobenzene (11, 14). Due to this fast reaction it is hypothesized that nitrosobenzene does not leave the enzyme and through the consumption of another NADH, nitrosobenzene is reduced to hydroxylaminobenzene (11).

1)

E + NADH E + S1

E---NADH ES1 EH ----ArNO2 E1S 2

EH E1 E + ArNO E+P

2)

EH - + ArNO2 E1 + S 2

Figure 7: Possible mechanism for NB reduction: Ping Pong mechanism. (Modified from (1)). 1): The first substrate (NADH) binds to the enzyme and reduces it, conformational change occurs. 2): The reduced enzyme is now able to bind the second substrate (NB). After the reduction of NB and production of nitrosobenzene, the enzyme returns to the beginning state.

Summarizing during the reduction of NB an N-O bond is broken. The N atom and the O atom will undergo to a primary isotope effect. Since we assume that elementary reaction step 4 in fig. 6 is the rate limiting step, the primary isotope effect will be measurable. Indicative substantial AKIE values for the cleavage of a N-O bond are about 1.03 for nitrogen and 1.04 for oxygen (4).

3.3. Expected bulk enrichment factor for NB

Using equation 3 it is possible to calculate the enrichment factors (!E) for atom E. Due to the different enzymatic reaction mechanisms, different dilution factors are expected.

3.3.1. NB oxidation
Parameters: C-C double bond transformation to C-C single bond: Dilution factor: 3 ($-value: 6/2) AKIEC=1.01(4) C 10 0 00 C-N cleavage: Dilution factor for element C: 6 ($-value:6) AKIEC=1.03 (4) C 30 0 00 Dilution factor for element N: 1 ($-value:1) AKIEN=1.03 (4) N 30 0 00 9

Term paper, June 2007 Enrichment factor for carbon (!C): Case 1: nitrite is released during the addition of oxygen 1 & 2# C 30 0 00 + $ 10 0 00 ! 8 0 00 6 % 6"

Case 2: nitrite is released after the addition of oxygen 2 C 10 0 00 3 0 00 6 Enrichment factor for nitrogen (!N): Case 1: nitrite is released during the addition of oxygen 1 N 30 0 00 30 0 00 1 Case 2: nitrite is released after the addition of oxygen N = 0

3.3.2. NB reduction
Parameters: N-O bond cleavage: Dilution factor for element N: 1 ($-value:1) AKIEN=1.03 (4) N 30 0 00 Dilution factor for element O: 2 ($-value:2) AKIEO=1.04 (4) O 40 0 00
Enrichment factor for nitrogen (!N):
1 1 Enrichment factor for oxygen (!O): 1 O 40 0 00 20 0 00 2

N 30 0 00 30 0 00

4. Conclusion
The results of this review show that a determination of the fate of NB in contaminated areas with CSIA should be possible. Different elements are involved in the oxidation and reduction reaction. !C is expected to be between -8 0/00 and -3 0/00 for the oxidative pathway and 0 0/00 for the reductive pathway. !N is expected to be about -300/00 for the reductive pathway. It is not possible to predict if nitrogen will be enriched in the oxidative pathway. If it is the case, the !N value will be also of 300/00. Oxygen is enriched in reductive pathway; unfortunately nowadays it is not possible to measure its isotopic signature with certainty. Therefore carbon is the best candidate for the determination of the pathway.

10

Term paper, June 2007

5. References
1.
Anusevicius, Z., A. Soffers, N. Cenas, J. Sarlauskas, M. Martinez-Julvez, and I. Rietjens. 1999. Quantitative structure activity relationships for the electron transfer reactions of Anabaena PCC7119 ferredoxin-NADP(+) oxidoreductase with nitrobenzene and nitrobenzimidazolone derivatives: mechanistic implications. Febs Letters 450:44-48. Boyd, D. R., and T. D. H. Bugg. 2006. Arene cis-dihydrodiol formation: from biology to application. Organic & Biomolecular Chemistry 4:181-192. Cenas, N., Z. Anusevicius, D. Bironaite, G. I. Bachmanova, A. I. Archakov, and K. Ollinger. 1994. The Electron-Transfer Reactions of Nadph-Cytochrome P450 Reductase with Nonphysiological Oxidants. Archives of Biochemistry and Biophysics 315:400-406. Elsner, M., L. Zwank, D. Hunkeler, and R. P. Schwarzenbach. 2005. A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environmental Science & Technology 39:6896-6916. Friemann, R., M. M. Ivkovic-Jensen, D. J. Lessner, C. L. Yu, D. T. Gibson, R. E. Parales, H. Eklund, and S. Ramaswamy. 2005. Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase. Journal of Molecular Biology 348:1139-1151. Hartenbach, A., T. B. Hofstetter, M. Berg, J. Bolotin, and R. P. Schwarzenbach. 2006. Using nitrogen isotope fractionation to assess abiotic reduction of nitroaromatic compounds. Environmental Science & Technology 40:7710-7716. Hofstetter T., H. A., Tobler N. and Bolotin J. 2007. Labguide on the course CompoundSpecific Isotope Analysis (CSIA). ETH Zrich. Hughes J.B., K. H. J., Nishino S.F., Spain J. C., and Zhongqi H. . 2000. Biodegradation of nitroaromatic compounds and explosives. Chapter 2, Strategies for Aerobic Degradation of Nitroaromatic Compounds by Bacteria: Process Discovery to Field Application. Hughes J.B., K. H. J., Spain J. C. 2000. Biodegradation of nitroaromatic compounds and explosives. Chapter 1, Introduction. Ju, K. S., and R. E. Parales. 2006. Control of substrate specificity by active-site residues in nitrobenzene dioxygenase. Applied and Environmental Microbiology 72:1817-1824. Koder, R. L., and A. F. Miller. 1998. Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1387:395-405. Lee, K. S., J. V. Parales, R. Friemann, and R. E. Parales. 2005. Active site residues controlling substrate specificity in 2-nitrotoluene dioxygenase from Acidovorax sp strain JS42. Journal of Industrial Microbiology & Biotechnology 32:465-473. Lessner, D. J., G. R. Johnson, R. E. Parales, J. C. Spain, and D. T. Gibson. 2002. Molecular characterization and substrate specificity of nitrobenzene dioxygenase from Comamonas sp strain JS765. Applied and Environmental Microbiology 68:634-641. Nishino, S. F., and J. C. Spain. 1993. Degradation of Nitrobenzene by a PseudomonasPseudoalcaligenes. Applied and Environmental Microbiology 59:2520-2525. Nishino, S. F., and J. C. Spain. 1995. Oxidative Pathway for the Biodegradation of Nitrobenzene by Comamonas Sp Strain Js765. Applied and Environmental Microbiology 61:2308-2313. Nivinskas, H., S. Staskeviciene, J. Sarlauskas, R. L. Koder, A. F. Miller, and N. Cenas. 2002. Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H : nitroreductase: quantitative structure-activity relationships. Archives of Biochemistry and Biophysics 403:249-258. Parales, R. E., R. Huang, C. L. Yu, J. V. Parales, F. K. N. Lee, D. J. Lessner, M. M. Ivkovic-Jensen, W. Liu, R. Friemann, S. Ramaswamy, and D. T. Gibson. 2005.

2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17.

11

Term paper, June 2007 Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygease enzyme systems. Applied and Environmental Microbiology 71:3806-3814. Rene P. Schwarzenbach, P. M. G., Dieter M. Imboden. 2003. Environmental Organic Chemistry, Chapter 13: Chemical Transformation I: Hydrolysis and Reactions Involving Other Nucleophilic Species. Schenzle, A., H. Lenke, J. C. Spain, and H. J. Knackmuss. 1999. Chemoselective nitro group reduction and reductive dechlorination initiate degradation of 2-chloro-5-nitrophenol by Ralstonia eutropha JMP134. Applied and Environmental Microbiology 65:2317-2323. Somerville, C. C., S. F. Nishino, and J. C. Spain. 1995. Purification and Characterization of Nitrobenzene Nitroreductase from Pseudomonas Pseudoalcaligenes Js45. Journal of Bacteriology 177:3837-3842.

18. 19. 20.

12